Electro-optical nucleic acid-based sensor array and method for detecting analytes

ABSTRACT

The present invention is directed to methods of detection, identification and monitoring of vapor phase analytes by using sensor arrays comprising fluorophore labeled nucleic acids, dried onto a substrate which react with vapor phase analytes. Methods of using and preparing such sensor arrays are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the U.S. Provisional PatentApplication Ser. No. 60/428,869, filed Nov. 25, 2002, and a co-pendingU.S. patent application Ser. No. 10/303,548 filed Nov. 25, 2002.

GOVERNMENT SUPPORT

The invention described herein was supported in part with U.S.Government funding under Defense Advance Research Projects AgencyContract No. DAAK60-97-K-9502, Office of Naval Research Grant No.N00014-95-1-1340, and National Institutes of Health Grant DC00228. TheU.S. government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to compositions and systemsuseful in monitoring of chemical hazards, air quality, and medicalconditions, and detecting explosives, mines, and hazardous chemicals.The invention provides nucleic acid-based sensors and methods fordetecting analytes. More particularly, the invention relates to nucleicacid-based optical sensors, sensor arrays, sensing systems and sensingmethods for sensing and detection of unknown analytes in vapor phase byway of real-time feedback and control of sampling conditions includingremote controlled systems and methods of making such sensors and sensorarrays.

BACKGROUND OF THE INVENTION

The serious threat of explosive, chemical and/or biological attacks posea particular challenge for national security in the current “postSeptember 11th, 2001” era. A method that could detect a wide range ofcompounds, and that could also be automated and remotely controlled andthat could be used in field conditions including airport, seaport, orother screening systems, would be particularly desirable. For example,currently only about 2% of all the containers are screened by any meansthat come through the seaports to the United States, because there areno suitable reliable, fast, easy and relatively cheap screening methodsavailable. For national security, it is imperative to develop screeningmethods that could detect, for example, explosives and toxic chemicalsthat may be transported into the United States. Detection methods foridentifying trace amounts of volatile compounds from, for example,explosives or chemical warfare agents, would be one possible way toapproach such novel screening methods for national security purposes.

Moreover, there are a number of other current and potential uses fordetection and identification of volatile compounds. For example,different chemical analyses have been used to detect the presence orabsence of a known target chemical in clinical diagnoses, to identifyunknown compounds and mixtures in basic research and drug discovery, andto document the identity and purity of known compounds, e.g., in testingand quality control in drug manufacturing processes. In addition tolaboratory analyses, chemical detection is also important outside of thelaboratory. Examples include bedside diagnoses, and environmentalmonitoring for hazardous materials. The “field” applications, includingdetection of explosives and chemical warfare agents, require small,portable, reliable, easy-to-use, inexpensive devices.

There are number of methods currently available for chemical analysis,each appropriate for a particular application and each having its ownstrengths and weaknesses. Examples include the various forms ofchromatography, including gas chromatography (GC), high performanceliquid chromatography (HPLC), and spectroscopy, including massspectroscopy (MS), ion mobility spectroscopy (IMS), Raman spectroscopyand infrared spectroscopy, as well as other chemical, immunological, andgravimetric methods. Also, combinations of different methods can providea powerful means of identifying unknown compounds, e.g., GC/MS which isused extensively in analytical chemistry laboratories.

A common feature of these analytical methods is that the chemical sampleneeds to be prepared prior to analysis. Liquid and solid samples areusually dissolved into an appropriate solvent. For analysis ofvapor-phase chemicals, a preconcentration step is often required toincrease the quantity of material for analysis.

Preconcentration of vapor-phase chemicals involves passing a largevolume of air over an adsorbent Tenax or solid phase microextraction(SPME) trap. The sample is removed from the trap using a small amount ofliquid solvent or is thermally desorbed directly into the input of a GCfor analysis (Zhang, Z., Yang, M. J., and Pawliszyn, J. (1994) Anal.Chem., 66:844A-853A).

Preconcentration followed by GC or GC/MS has been used to detect andquantify volatile chemicals in a variety of studies with relevance tohealth care and domestic security, for example, detection ofcontaminants in water (Lambropoulou, D. A. and Albanis, T. A. (2001) J.Chromatogr., 922:243-255; Cancho, B., Ventura, F., and Galceran, M. T.(2002) J. Chromatogr., 943:1-13) and soils (Cam, D. and Gagni, S. (2001)J. Chromatogr. Sci., 39:481-486), detection of toxic substances in blood(Bouche, M. P., Lambert, W. E., Bocxlaer, J. F. V., Piette, M. H., andLeenheer, A. P. D. (2001) J. Anal. Toxicol., 26:35-42;Musshoff, F.,Junker, H., and Madea, B. (2002) J. Chromatogr. Sci., 40:29-34),detection of drugs in postmortem tissue (Mosaddegh, M. H., Richardson,R., Stoddart, R. W., and McClure, J. (2001) Ann. Clin. Biochem.,38:541-547), detection of organic compounds in normal breath (Grote, C.and Pawliszyn, J. (1997) Anal. Chem., 69:587-596) and in the breath oflung cancer patients (Phillips, M., Gleeson, K., Hughes, J. M. B.,Greenberg, J., Cataneo, R. N., Baker, L.; and McVay, W. P. (1999)Lancet, 353:1930-1933), characterization of explosive signatures(Jenkins, T. F., Leggett, D. C., Miyares, P. H., Walsh, M. E., Ranney,T. A., Cragin, J. H., and George, V. (2001) Talanta, 54:501-513), anddetecting Sarin in air and water (Schneider, J. F., Boparai, A. S., andReed, L. L. (2001) J. Chromatogr. Sci., 39:420-424). For rapid detectionof volatile compounds, it would be advantageous to avoid specific samplepreparation steps. This would be especially desirable in applicationswhere detection is performed in field conditions, outside a laboratory.

Volatile chemical analyses using these methods require optimizations foreach analysis problem. For example, the GC column, GC detector, trapcoatings, and flow rates all need to be optimized for particularvolatiles of interest. In addition, preconcentration can takeconsiderable time to collect sufficient material in the trap. The timerequired depends on the sorbent coating on the trap (different Tenaxcoatings have different affinities for different chemical compounds) andon the original concentration of sample in the air. Such analyticalmethods are therefore generally inappropriate for rapid analyses, suchas security screening, real-time environmental monitoring, or bedsidediagnoses. Therefore, it would be advantageous to develop a detectionsystem that is capable of rapidly analyzing a wide array of differentcompounds in varying concentrations.

For air sampling, ah alternative to preconcentration consists of systemscontaining dedicated sensors that are responsive to particular compoundsof interest. Common examples include home detectors for carbon monoxide,propane, and natural gas. Although sensors are available that arebroadly responsive, e.g., sensors that respond to many volatile organiccompounds, these devices do not identify the vapor detected. While asystem containing a dedicated selective sensor can respond rapidly toits cognate analyte and may not require preconcentration, the ability todetect and identify multiple volatile compounds would require a separatesensor selective for each compound of interest. Further, such methodspreclude detection of future compounds of interest. Therefore, it wouldbe desirable to develop a system that is capable of sensing as well asidentifying a wide range of compounds.

For detecting, discriminating, and identifying volatile compounds in theair, one of the most highly developed chemical detection devices isarguably the olfactory system of terrestrial animals. Olfactoryabilities include high sensitivity (Passe, D. H. and Walker, J. C.(1985) Neurosci. Biobehav. Rev., 9:431-467), the ability to detect anddiscriminate many different compounds (e.g., Youngentob, S. L., Markert,L. M., Mozell, M. M., and Homung, D. E. (1990) Physiol. Behav.,47:1053-1059; Slotnick, B. M., Kufera, A., and Silberberg, A. M. (1991)Physiol. Behav., 50:555-561; Lu, X.-C. M., Slotnick, B. M., andSilberberg, A. M. (1993) Physiol. Behav., 53:795-804), and the abilityto make fine odorant discriminations (e.g., individual recognition inrodents—Yamazaki, K., Singer, A., and Beauchamp, G. K. (1998-1999)Genetica, 104:235-240). Numerous mechanisms influence these capabilitiesat points in the process even before odorant molecules interact withreceptor proteins. Sniffing behavior, nasal aerodynamics, mucussolvation, and odorant clearing all likely play a role in olfactoryabilities (Christensen, T. A. and White, J. (2000). Representation ofolfactory information in the brain. In Finger, T. E., Silver, W. L., andRestrepo, D., editors, Neurobiology of Taste and Smell, pages 201-232.John Wiley & Sons, New York). Once odorants reach the olfactory receptorproteins in the nasal mucosa, the system does not devote one receptorprotein to each individual odorous ligand. Rather, even single compoundsinteract with many broadly-responsive receptor proteins, producingwidespread spatiotemporal patterns of activity in the olfactory sensoryneuron population—in other words, activity in many sensor elements thatevolve over time (MacKay-Sim, A., Shaman, P., and Moulton, D. G. (1982)J. Neurophysiol., 48:584-596; Kent, P. F. and Mozell, M. M. (1992) JNeurophysiol, 68:1804-1819). These patterns of activity are theninterpreted by parallel processing elements in the olfactory areas ofthe brain, producing widespread activation in the neuronal populationsat each level of the olfactory pathway (for reviews, see Kauer, J. S.(1987). Coding in the olfactory system. In Finger, T. E. and Silver, W.L., editors, Neurobiology of Taste and Smell, pages 205-231. John Wiley& Sons, Inc, New York; Kauer, J. s. (1991). Trends Neurosci, 14:79-85;Christensen, T. A. and White, J. (2000). Representation of olfactoryinformation in the brain. In Finger, T. E., Silver, W. L., and Restrepo,D., editors, Neurobiology of Taste and Smell, pages 201-232. John Wiley& Sons, New York).

The properties of the olfactory system suggest that engineered devicesbased on olfactory mechanisms may have advantages for detecting andidentifying volatile compounds. Such a device, called an “artificialnose” or “electronic nose,” was first described in the early 1980's(Persaud, K. and Dodd, G. (1982). Nature, 299:352-355), and a number ofsystems have been reported since then (see, e.g., Grate, J. W.,Rose-Pehrsson, S. L., Venezky, D. L., Klusty, M., and Wohltjen, H.(1993) Anal. Chem., 65:1868-1881; Freund, M. S. and Lewis, N. S. (1995).Proc. Nat. Acad. Sci. USA, 92:2652-2656; White, J., Kauer, J. S.,Dickinson, T. A., and Walt, D. R. (1996) Anal. Chem., 68:2191-2202).

All of these devices incorporate the two main features that define anelectronic nose: 1) an array of broadly-responsive sensors and 2) apattern recognition method for processing sensor output. Like in theolfactory system, odorants interact with multiple sensors, producing apattern of activation across the array. Commercial and researchelectronic noses use a variety of technologies for chemical sensingincluding conducting polymers, surface acoustic wave devices,solid-state devices, and optical interrogation. Pattern recognitionmethods generally involve statistical methods or computational neuralnetworks (for reviews, see Gardner, J. W. and Bartlett, P. N., editors(1992). Sensors and sensory systems for an electronic nose. KluwerAcademic Publishers, Dordrecht, The Netherlands; Gardner, J. W. andBartlett, P. N. (1994). Sensors and Actuators B, 18-19:211-220; Gardner,J. W. and Hines, E. L. (1997). Pattern analysis techniques. InKress-Rogers, E., editor, Handbook of Biosensors and Electronic Noses:Medicine, Food, and the Environment, pages 633-652. CRC Press,BocaRaton, Fla.; Dickinson, T. A., White, J., Kauer, J. S., and Walt, D.R. (1998). Trends Biotechnol., 16:250-258).

Potential and actual uses of commercial electronic noses includefood/beverage analysis, environmental monitoring, and medical diagnosis(Ping, W., Yi, T., Haibao, X., and Farong, S. (1997) Biosens.Bioelectron., 12:1031-1036; Dickinson, T. A., White, J., Kauer, J. S.,and Walt, D. R. (1998) Trends Biotechnol., 16:250-258; Aathithan, S.,Plant, J. C., Chaudry, A. N., and French, G. L. (2001) J. Clin.Microbiol., 39:2590-2593).

Vapor phase chemical detection systems based on arrays ofbroadly-responsive sensors offer a number of potential advantages overtraditional analytical devices. An electronic nose directly samples theair, so no sample preparation is necessary. The time required fordetection is limited only by the time required for the chemical sensorsto respond and for the pattern recognition calculation, which is fastusing modem computer technology. With rapidly-responding sensors, rapiddetection of volatiles is therefore possible. In addition, whiletraditional analytical instruments tend to be large and requireconsiderable power, sensor array devices have the potential for beingsmall and portable. Although handheld IMS devices are available, theyare currently tuned to specific, restricted tasks, such as use of theIontrack Instruments VaporTracer2 for explosives or drugs, and thereforelack the broad-band nature of an electronic nose.

Sensor array devices would also have a number of advantages over systemsusing mono-specific sensors. First, truly “mono-specific” sensors aredifficult (if not impossible) to produce; broadly-responsive sensors canbe readily made. Second, even if mono-specificity could be achieved,detection of several compounds would require development of a separatesensor for each compound of interest. Conversely, a relatively smallarray of broadly-responsive sensors is theoretically capable ofdiscriminating a large number of different compounds (Alkasab, T. K.,White, J., and Kauer, J. S. (2002) Chem. Senses, 27:261-275). Third, adevice containing sensors specific for a finite number of compounds isincapable of detecting any others outside its defined target set. Adevice containing broadly-responsive sensors would have the potentialfor detecting and discriminating compounds of future interest.

It would be advantageous to develop sensors capable of detecting andcorrectly identifying a large range of analytes, e.g., volatilechemicals. Such sensors would be particularly useful in domesticsecurity applications, such as detecting explosives and chemical warfareagents.

SUMMARY OF THE INVENTION

We have, surprisingly, discovered that nucleic acids with attachedfluorophores and dried onto a substrate react with volatile chemicalcompounds or analytes in ambient air and can therefore be used assensors to detect analytes in the air that react thereto. This isdistinctly different from other nucleic acid-based sensing materialsthat work only when both the analytes and nucleic acid materials arepresent in aqueous solution.

The term “analyte” as referred to throughout the specification refers toany molecule or compound. A “volatile analyte” refers to a molecule orcompound in gaseous or vapor phase, that is present, for example, in theheadspace of a liquid, in ambient air, in a breath sample, in a gas, oras a contaminant in any of the foregoing. Analytes further includesolid-phase compounds that are small enough to remain suspended in air,e.g., dust, molecules and compounds-present on the surfaces of particlespresent in gaseous or vapor phase, such as virus envelope proteins orbacterial cell surface or spore surface molecules, macromolecules thatare cast off from other sources such as DNA, RNA, and proteins.

Accordingly, the present invention provides a nucleic acid-basedchemical sensor, sensing system and sensing and identification methodwhich provide for a nucleic acid-based multi-sensor, cross-reactive,sensor array having a rapid response time, a rapid sampling time,dynamic modulation of sampling and detection parameters, intelligentfeedback control of analyte sampling conditions, smart mode sampling,smart detection through application of sophisticated analyte detectionalgorithms, high throughput screening of sensors, and high sensitivity,discrimination, and detection capability for a variety of targetanalytes.

The invention further provides a nucleic acid-fluorphore-based analytesensing system which can transmit identifying information on variousodors or smells, e.g., vapor or gaseous analytes, remotely, for example,over the Internet, or via a wireless communication system.

In one embodiment, the present invention provides a method for detectingand/or identifying an analyte, e.g., a volatile analyte, in an airsample comprising the steps of:

-   -   a) contacting said air sample with a nucleic acid-based sensor        array comprising a substrate and a nucleic acid labeled with        (attached to) a fluorophore dispersed on the substrate, said        nucleic acid labeled with a fluorophore providing a        characteristic optical response when subjected to excitation        light energy in the presence of the analyte; and    -   b) detecting the presence or absence of the analyte.    -   c) identifying the analyte found in the air sample.

The substrate can be fabricated of different materials, including, forexample, papers, fiberglass, silk, and fabrics made of syntheticmaterials.

In one preferred embodiment, the nucleic acid/fluorophore is dispersedon a plurality of internal and external surfaces within the substrate.

In one embodiment, contacting is accomplished by drawing an air samplesuspected to contain the analyte into a sample chamber and exposing thearray to the air sample. In a preferred embodiment, the air sample isdrawn through the chamber for no more than five seconds.

The detecting may be accomplished by illuminating said sensor withexcitation light energy and measuring an optical response produced bythe sensor due to the presence of said volatile compound with a detectormeans. Detector means include, for example, a variety of photodetectorssuch as photomultiplier tubes (PMTs), charge-coupled display device(CCD) detectors, photovoltaic devices, phototransistors, andphotodiodes. In a preferred embodiment, filtered photodiode detectorsare used.

In all embodiments, the analyte can be identified by employing apattern-matching algorithm and comparing the optical response of thenucleic acid-based sensor array with the characteristic opticalresponse.

In specific embodiments, the analyte can be identified by measuring thespatio-temporal response patterns of the optical response andrecognizing the patterns through a method selected from templatematching, neural networks, delay line neural networks, or statisticalanalysis. The air sample may be suspected of containing analytes from avariety of substances, including explosive materials or chemical weaponsagents.

The present invention flrther provides a sensing system for detectingand identifying an analyte in an ambient air sample. The systemincludes: a) a nucleic. acid-based sensor array comprising a pluralityof nucleic acids; b) a fluorophore attached to the nucleic acids; c) aplurality of substrates wherein the nucleic acids with fluorophore areattached to; d) a substrate support; e) an excitation light source arrayincluding a plurality of light. sources optically coupled to the sensorelements; f) a detector array comprising a plurality of detectorsoptically coupled to said sensor elements; g) a sample chamber forhousing the sensor elements, the light source array, and the detectorarray; h) a sampling means attached to the chamber for drawing theambient air into the chamber for contact with the sensor array for acontrolled exposure time; i) a controller means in electricalcommunication with the light sources, the detectors, and the samplingmeans, the controller means electrically coordinating and switching thesampling means with the light sources and the detectors for sampling theambient air, measuring optical responses of the array sensors to theambient air sample, and detecting the volatile compound; and j) ananalyte identification algorithm for comparing the measured sensoroptical responses to characteristic optical responses of the sensors totarget analytes and identifying the analyte in the ambient air sample.

The elements of the analyte sensing system may be used together in ahand-held device, a device attached to another object, e.g., a shippingcontainer, or used in conjunction with another screening device such asan x-ray screening machine. Alternatively, separate elements of thesystem, e.g., elements a)-i), can be used as one or more sensing units,while the analyte identification algorithm resides on a computer at aremote or separate location. One or more sensing units can be connectedwith the computer via a wired or wireless network.

In another preferred embodiment the identification algorithm reports adetection event when the sensor responses are different from blank airand identifies the analyte present using a pattern-match algorithm.

In one preferred embodiment, the system comprises one or more remotesensing units of the analyte sensing system with nucleicacid-fluorophore sensor arrays wirelessly connected to each others andthe unit with the analyte identification algorithm, so that theinformation about the analytes is transferred to a remote location

Therefore, in one embodiment, the invention provides a sensor arraysysiem for remote characterization of a gaseous or vapor sample,comprising: a) a plurality of sensors, wherein at least one sensorcomprises nucleic acid/fluorophore combination comprising a plurality ofnucleic acids attached to a fluorophore, wherein the plurality ofsensors provide a detectable signal when contacted by an analyte; b) ameasuring apparatus, in communication with plurality of sensors capableof measuring the detectable signal; c) a transmitting device, incommunication with the measuring apparatus for transmitting informationcorresponding to the detectable signal to a remote location via theInternet, fiber optic cable, and/or an air-wave frequency; and acomputer comprising a resident algorithm capable of characterizing theanalyte.

The invention further provides a method of selecting nucleic acidscapable of responding to a vapor phase analyte, said method comprising:a) contacting the nucleic acid labeled with a fluorophore with ananalyte in vapor phase; and b) measuring the emission proflile of thefluorophore in the presence and absence of the target analyte, wherein adifference in the emission profile indicates that the nucleic acid isresponsive to the analyte in vapor phase.

BRIEF DESCRIPTION OF THE FIGURES

This invention is pointed out with particularity in the appended claims.Other features and benefits of the present invention can be more clearlyunderstood with reference to the specification and the accompanyingdrawings in which:

FIGS. 1A and 1B show an example of a hand-held configuration of theElecto-Optical Vapor Interrogation Device (EVID). FIG. 1A shows aschematic view of the EVID sensor chamber, air flow path (30) (thickarrows), signal pathways (solid arrows), and computer control lines(dashed arrows) (8). The 3-way valve for switching between odorous andclean air is implemented as a pair of servo controlled valves (10). InFIGS. 1A and 1B the following parts are shown: panel of light emittingdiode light sources (12) and excitation filters (28); the panel ofphotodiode detectors (26) and emission filters; (14); sniff pump (4);control and feedback control (double arrow in two directions) (16);computer (18); 16 channel integrating amplifiers and 20 bit A/D/converters (20); inhale path (22) clean air from the source (24). FIG.1B outlines a top view of the same system with LEDs (12); emissionfilter (14) excitation filter (28); and photodiodes (26).

FIGS. 2A and 2B show temporal responses of sensor made from YO-PRO andpBluescriptSK DNA. FIG. 2A shows a sensor made from YO-PRO, then rinsedin 70% ethanol for 5 min. FIG. 2B shows a sensor made from YO-PRO and 5ng total pBlueScriptSK DNA. Analyte dilutions as fractions of saturatedvapor were: Water, 10⁻¹; methanol (MeOH), 10⁻¹; triethylamine, 10⁻²; andpropionic acid, 10⁻¹. Each trace represents the mean of 10presentations; error bars indicate +/−1 S.D. For experiments withDNA-based analyte sensors, similar methods were used for each type ofsensor. Briefly, DNA in solution was diluted to the desiredconcentration (0.2-40 ng/μl) in TE (1 Tris, 0.5 mM EDTA). 20 μl ofdilute DNA was mixed with 1 μl concentrated dye stock and incubated atroom temperature for 5 minutes. Dye-only controls were made of 1 μl dyestock in 20 μl TE. Sensors were made on a substrate of acid-washed 16xxsilkscreen (10 mm×12 mm). DNA/dye mixtures were pipetted onto thesubstrate and allowed to dry for 25 minutes. Each sensor was rinsed in70% ethanol for 5 minutes, allowed to dry, then attached to supports onglass coverslips for testing in the EVID (FIG. 2B).

FIGS. 3A and 3B show temporal responses of sensors made from differentshort sequences of single-stranded DNA and OliGreen dye. FIG. 3A showsan oligomer DS003, which has the sequenceGATCCTTGCTACCCTCTCCTAGGAACGATGGGA (SEQ ID NO: 5). FIG. 3B shows anoligomer AJ001, which has the sequence ACCAGGACCTGACTAAGCAGAT (SEQ IDNO: 4). See FIG. 2 for sensor construction details and analytedilutions. Each trace represents the mean of 10 replicates; error barsindicate +/−1 S.D.

FIGS. 4A and 4B show analyte concentration responses of twooligonucleotides labeled with the fluorescent dye Cy3(tm) duringsynthesis (using Cy3(tm) phosphoramidite from Glen Research). FIG. 4Ashows LAPP1, which is the sequence GAGTCTGTGGAGGAGGTAGTC (SEQ ID NO: 1).FIG. 4B shows LAPP2, which is the sequence CTTCTGTCTTGATGTTTCTCAACC (SEQID NO.:2). The oligomers were stored in Tris-NaCl (10 mM Tris, 50 mMNaCl, pH 8) at 225 ng/μl, then diluted to a concentration of 50 ng/μl indistilled water just before use. See FIG. 2 for sensor constructiondetails. Signal amplitudes are the parameters resulting from theexponential fit of the sensor temporal signals as described below.Sensor signals and data processing. Each data point is the mean of 10presentations; error bars indicate +/−1 S.D.

FIG. 5 shows an overview of steps for sensor library creation andscreening. The PCR template a) or primer extension template b) isamplified with two or one primer(s), respectively. Step 1: Synthesizerandom sequence library; Step 2: Dilute library; Step 3: Put samplesinto 104 96-well plates; Step 4: Amplify and label the nucleic acids;Step 5: Create high-density sensor library using a robotic spotter; Step6: Image sensor library with array scanner before and after applying thevapor phase analytes.

FIG. 6 shows an example of a strong propionic acid odor response in oneset of sensor spots. Data were collected using a ScanArray 4000microarray scanner. The image on the left shows the backgroundfluorescence of the sensor spots in clean air. The center image showsthe fluorescence levels in the same spots after saturated vaporpropionic acid was injected into the test chamber. The image on theright shows the change in fluorescence when the image on the left wassubstracted from the image in the middle. Arrays of spots were appliedto the coverslip in blocks of 12×12 (12 replicates vertically and 12different sequences horizontally); two replicate blocks (Rep 1 and Rep2) were applied under three different ionic conditions: 50 MM MgCl₂, 50mM NaCl, and water. One sensor sequence, TLAPP1 in water, showed astrong increase in fluorescence, other sequences showed smaller changesin fluorescence.

FIGS. 7A and 7B show a diagrams of an exemplary chamber (32) fordelivering analytes to a sensor array when testing the nucleic acids fortheir responsiveness to analytes in vapor phase. FIG. 7A shows a topview of the chamber and FIG. 7B shows a side view of the chamber. Solidblack (34) indicates stainless steel, darker grey (36) indicates 40micron pore size stainless steel filter.

The tube wherein the odor is injected in is a 21 gauge Teflon tubing andis indicated with a white tube (38). The analyte is injected into thetube and comes out through the filter (40) (dark grey block). Theinterior chamber (42) contains the coverslip which is exposed to theanalyte after the analyte is passed through the filter (white areainside the stainless steel walls of the chamber). The dimensions ofchamber shown in this figure are appropriate for reading the glasscoverslip with a ScanArray 4000.

FIG. 8 shows a block diagram of a sensor system of the present inventionwherein the analysis is performed in a remote location showing thesensor chamber (1) with nucleic acid arrays (2) inside the chamber.

FIG. 9 is a block diagram showing hardware components of one embodimentof the system.

FIG. 10 is a schematic diagram of a sensor array module.

FIG. 11 is the sequence of oligomers with random internal sequence andfixed ends.

FIG. 12 shows PCR reaction primers and double stranded product. Theasterisk represents Cy3(tm) labeling of the 5′ dTTP nucleotide of thelower primer.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid-based sensing method and sensing device design of thepresent invention mimics and parallels the structure and operationalcharacteristics of the mammalian olfactory system through thecombination of electro-optical hardware component modules,microprocessor control and software sampling and detection algorithms.The sample cavity design mimics the mammalian nasal cavity where odorsor smells (i.e. vapor analytes) are drawn into the sensing module(“sniffed” or “inhaled”) and their interaction with a plurality ofsensing elements (“sensory neurons”) in a sensor array triggers anexternal event.

Analyte applications include broad ranges of chemical classes such asorganics including, for example, alkanes, alkenes, alkynes, dienes,alicyclic hydrocarbons, arenes, alcohols, ethers, ketones, aldehydes,carbonyls, carbanions, biogenic amines, thiols, polynuclear aromaticsand derivatives of such organics, e.g., halide derivatives, etc.,biomolecules such as sugars, isoprenes and isoprenoids, fatty acids andderivatives, etc.

Accordingly, commercial applications of the sensors, arrays and nosesinclude environmental toxicology and remediation, biomedicine, materialsquality control, food and agricultural products monitoring, anaestheticdetection, breath alcohol analyzers, hazardous spill identification,explosives detection, fugitive emission identification, medicaldiagnostics, fish freshness, detection and classification of bacteriaand microorganisms both in vitro and in vivo for biomedical uses andmedical diagnostic uses, monitoring heavy industrial manufacturing,ambient air monitoring, worker protection, emissions control, productquality testing, leak detection and identification, oil/gaspetrochemical applications, combustible gas detection, H₂S monitoring,hazardous leak detection and identification, emergency response and lawenforcement applications, illegal substance detection andidentification, arson investigation, enclosed space surveying, utilityand power applications, emissions monitoring, transformer faultdetection, food/beverage/agriculture applications, freshness detection,fruit ripening control, fermentation process monitoring and controlapplications, flavor composition and identification, product quality andidentification, refrigerant and fumigant detection,cosmetic/perfume/fragrance formulation, product quality testing,personal identification, chemical/plastics/pharmaceutical applications,leak detection, solvent recovery effectiveness, perimeter monitoring,product quality testing, hazardous waste site applications, fugitiveemission detection and identification, leak detection andidentification, perimeter monitoring, transportation, hazardous spillmonitoring, refueling operations, shipping container inspection,diesel/gasoline/aviation fuel identification, building/residentialnatural gas detection, formaldehyde detection, smoke detection, firedetection, automatic ventilation control applications (cooking, smoking,etc.), air intake monitoring, hospital/medical anesthesia &sterilization gas detection, infectious disease detection and breathapplications, body fluids analysis, pharmaceutical applications, drugdiscovery, telesurgery, and the like.

Biogenic amines such as putrescine, cadaverine, and spermine are formedand degraded as a result of normal metabolic activity in plants, animalsand microorganisms and can be identified in order to assess thefreshness of foodstuffs such as meats (Veciananogues, J. Agr. FoodChem., 45:2036-2041, 1997), cheeses, alcoholic beverages, and otherfermented foods. Additionally, aniline and o-toluidine have beenreported to be biomarkers for subjects having lung cancer (Preti et al.,J. Chromat. Biomed. Appl. 432:1-11, 1988), breath ammonia in diagnosis,treatment assessment, and follow-up in hepatic encephalopathy (Shimamotoet al., Hepatogastroenterology, 47(32):443-5, 2000), while dimethylamineand trimethylamine have been reported to be the cause of the “fishy”uremic breath odor experienced by patients with renal failure.(Simenhoff, New England J. Med., 297:132-135, 1977). Thus, in generalbiogenic amines and thiols are biomarkers of bacteria, disease states,food freshness, and other odor-based conditions. Thus, the nucleicacid-fluorophore based nose sensor elements and arrays discussed hereincan be used to monitor the components in the headspace of urine, blood,sweat, and saliva of human patients, as well as breath, to diagnosevarious states of health, such as the timing of estrus (Lane et al., JDairy Sci 81(8):2145-50, 1998), and diseases as discussed herein. Inaddition, the sensor elements can be used for food quality monitoring,such as fish freshness (which involves volatile amine signatures), for.environmental and industrial applications (oil quality, water quality,air quality and contamination and leak detection), for other biomedicalapplications, for law enforcement applications (breathalayzers), forconfined space monitoring (indoor air quality, filter breakthrough,etc.) and for other applications delineated above to add functionalityand performance to sensor arrays through improvement in analytedetection by use in arrays that combine sensor modalities. Accordingly,the invention provides physicians and patients with a method to monitorillness and disease from remote locations. It is envisioned that thesystems of the invention will be useful in medical care personnelmonitoring patients who are bed-ridden at home or whom require continualmonitoring of a particular disease state. Such remote monitoring abilityeliminates the need for repeated trips to a doctors office or hospitaland can provide physicians with real-time data regarding a patient'shealth and well-being.

In one embodiment, analyte interaction with the nucleicacid/fluorophore-based sensing elements produces emitted light energy ata detectable characteristic wavelength when the sensor elements areilluminated by excitation light energy from a filtered LED array. Themulti-element nucleic acid-based sensor array of the present inventionthus mimics the sensory neurons of the olfactory system in responding tothe external triggering event, emitted light energy signaling thepresence of an analyte, and detecting this triggering event by way of afiltered photodiode array (“Detection”). The photodiode preamplifiersmimic an olfactory sensory neuron by converting the optical signal to anelectrical voltage signal (“Transduction”) which is amplified,manipulated and transported via electrical circuits (“Transmission”) toan analog-digital (“A/D”) converter and a software controlledmicroprocessor for data manipulation, analysis, feedback control,detection and identification (“Integration”). The Detection,Transduction, Transmission, and A/D features are replicated for eachnucleic acid-based sensor element in the array. The sensor array of thepresent invention may be expanded or contracted without limit by addingor removing elements and channels according to the requisite analytedetection, discrimination and identification needs of a specificsampling application.

FIGS. 1A and 1B provide an overview of the analyte sensing and detectionsystem of the present invention. Analytes from an odor source (2) aresniffed i.e. transported to the sensor array where the odors interactwith the array of nucleic acid/fluorophore-based sensor elements using a“sniff pump” (4). Light energy excitation of the sensor elements (6) inthe presence of the odors produces a detectable optical response signaldue to changes in emitted light produced by analyte interaction with thenucleic acid/fluorophore compounds in the sensor elements. Thespatio-temporal optical response of the nucleic acid-based array to theodor is detected, recorded, manipulated, and then matched to knowntarget odors via smart analytical algorithms, resident at computer 18,which apply, for example, pattern matching, neural network, neuronalnetwork, or statistical analysis methods to detect, discriminate andidentify the odor.

The hardware and software components and configuration of the nucleicacid/fluorophore-based sensor of the present invention provide for acompact, portable, inexpensive, expandable, rapidly responding sensingdevice that can modify its detection strategy on the fly. The design andmethod provides for real-time, on-the-fly, modulation of: a) the outputof light emitting diodes (LEDs), such as wavelength, intensity, andfrequency; b) the detection properties of photodiodes, such aswavelength, gain, and frequency; c) the sampling parameters, such asfrequency, duration, number, velocity, and rise-fall dynamics; and d)sampling time constant or temporal filter settings, for dynamicallyresponsive, smart feedback control in sampling, detection andidentification of analytes.

In addition to dynamic response modulation, the device and methodfurther provide for hardware and algorithm implementations whichevaluate the synchrony and noise characteristics across differentsensors, especially those of the same composition being examined atdifferent wavelengths. This provides a powerful tool for identifying andutilizing small response signals and rejecting noise.

By providing for independently illuminated, detected, recorded, andmodulated sensing channels, levels of flexibility, expandability,portability, efficiency, and economy are achieved that are difficult torealize with the currently existing odor detectors. In addition, the useof small, inexpensive, flexibly programmable, computationalmicrocomputer platforms and interchangeable nucleicacid/fluorophore-based sensors and sensor array modules provide forextreme flexibility and tailoring of sensor performance and capabilitiesto real world sensing applications, such as wirelessly connected sensingunits. One or more of such units can be placed, for example, in a tunnelthrough which objects can be directed and screened for odors oranalytes. Applications of such wireless systems comprising the nucleicacid-fluorophore sensor arrays of the present invention include, but arenot limited to screening for mail, screening for trucks or shipcontainers, cars, luggage and other such subject for odors andloranalytes.

An example of a wireless system useful according to the presentinvention is described in detail in, for example, U.S. Pat. No.6,631,333, incorporated herein by reference in its entirety.

Nucleic acids useful according to the present invention include singleand double-stranded RNA and single and double-stranded DNA and cDNA.Nucleic acid, oligonucleotide, and similar terms used herein alsoinclude nucleic acid analogs, i.e. analogs having other than aphosphodiester backbone. For example, the so-called peptide nucleicacids, which are known in the art and have peptide bonds instead ofphosphodiester bonds in the backbone are considered within the scope ofthe present invention (Nielsen et al. Science 254, 1497 (1991)).Alternatively, modified bases can be used in the nucleic acid sequence.

Examples of such modified bases are listed below on Table 1: TABLE 1Examples of Modified Bases Code Modified base ac4c 4-acetylcytidinechm5u 5 (carboxyhydroxymethyl)uridine cm 2′-O-methylcytidine cm5u 5-carbamoylmethyluridine cmnm 5s2u5-carboxymethylaminomethyl-2-thiouridine cmnm 5u5-carboxymethylaminomethyluridine d dihydrouridine fm2′-O-methylpseudouridine gal q beta,D-galactosylqueuosine gm2′-O-methylguanosine i-inosine i6a. N6-isopentenyladenosine m1a1-methyladenosine m1am 2′-O-methyl-1-methyladenosine m1f1-methylpseudouridine m1g 1-methylguanosine m1i 1-methylinosine m22g2,2-dimethylguanosine m22gm N2,N2,3′-trimethylguanosine m2a2-methyladenosine m2g 2-methylguanosine m3c 3-methylcytidine m5c5-methylcytidine m6a N6-methyladenosine m7g 7-methylguanosine mam5s2u5-methylaminomethyl-2-thiouridine mam5u 5-methylaminomethyluridine man qbeta,D-mannosylqueuosine mcm5s2u 5-methoxycarbonylmethyl-2-thiouridinemcm5u 5-methoxycarbonylmethyluridine mo5u 5-methoxyuridine ms2i6a2-methylthio-N6-isopentenyladenosine ms2t6aN-((9-beta-D-ribofuranosyl-2-methylthiopurin-6- yl)carbamoyl) threoninemt6a N-((9-beta-D-ribofuranosylpurine-6-yl)N-methyl- carbamoyl)threonine mv uridine-5-oxoacetic acid methylester o5uuridine-5-oxyacetic acid(v) osyw wybutoxosine p pseudouridine qqueuosine s2c 2-thiocytidine s2t 5-methyl-2-thiouridine s2u2-thiouridine s4u 4-thiouridine t 5-methyluridine t6aN-((9-beta-D-ribofuranosylpurine-6- yl)carbamoyl)threonine tm2′-O-methyl-5-methyluridine um 2′-O-methyluridine × 3-(3-amino-3-carboxypropyl)uridine,(acp3)U yw wybutosine

The length of the nucleic acid sequences can vary between about 1 baseof single stranded DNA up to about 3 thousand bases of double strandedDNA. Preferably about 18-24 base pair oligonucleotides are used.

Nucleic acids useful according to the present invention can besynthesized using methods well known to one skilled in the art. Forexample, a solid-phase phosphotriester approach can be used as describedin Sproat et al. (Solid-phase synthesis of oligodeoxyribonucleotides bythe phosphotriester method, in Oligonucleotide Synthesis—A practicalapproach (Gait, M. J., Ed.), IRL Press, Oxford pp. 83-115, 1984). Theconcept of the solid-phase phosphotriester approach has four basicaspects: the oligonucleotide is synthesized while attached covalently toa solid support, excess soluble protected nucleotides and couplingreagent can drive a reaction near to completion, the reaction is carriedout in a single reaction vessel to diminish mechanical losses due tosolid support manipulation, allowing synthesis with minute quantities ofstarting materials, and the heterogeneous reactions are standardized.All these procedures are easily automated and several commerciallyavailable oligonucleotide synthesizers are known to one skilled in theart. The most used chemical route for solid-phase oligonucleotidesynthesis is the phosphite triester method as modified by Beaucage andCaruthers (Beaucage, S. L., and Caruthers, M. H. (1981) Deoxynucleosidephosphoramidites—A new class of key intermediates fordeoxypolynucleotide synthesis, Tetrahedron Lett. 22,1859-1862).

Alternatively, nucleic acids can be isolated from libraries comprisingnucleic acid fragments in the form of, for example, plasmids, cosmids,yeast artificial chromosomes, and bacterial artificial chromosomes. Thenucleic acids can also be isolated from any other source such asviruses, and procaryotic or eucaryotic cells. Nucleic acid isolationmethods are routine and protocols can be found, for example fromMolecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and Russel,Cold Spring Harbor Laboratory Press, 2001.

After isolation, the isolated nucleic acids can be further modified, forexample, by restriction enzyme digestion. Isolated nucleic acids canalso be amplified using PCR and either random or specific primersequences. Such primer sequences can also be labeled with a fluorophoreduring the oligonucleotide synthesis.

In one embodiment, the nucleic acids useful in the present invention aresimilar to aptamers which can be selected from existing aptamers or fromrandom sequence libraries. Aptamers are defined as single-stranded ordouble-stranded nucleic acids which are capable of binding proteins orother small molecules with high specificity in aqueous solution. In thepresent invention, the nucleic acid-based sensors differ from aptamersin two important respects: 1) the nucleic acids sensors are dried onto asubstrate and interact directly with compounds in the air, and 2) thenucleic acid sequences are selected for their capacity to react, incombination with a fluorophore, to broad ranges of volatile compounds.Generally, aptamers are selected from a large number of non-interactingoligonucleotides and they originate from in vitro selection experimentstermed “SELEX” for systematic evolution of ligands by exponentialenrichment, that, starting from random sequence libraries, optimize thenucleic acids for high-affinity binding to given ligands (C. Tuerk andL. Gold, Science 249, 505 (1990); A. D. Ellington and J. W. Szostak,Nature 346, 818 (1990)). Reviews on in vitro selection and aptamers,see, e.g., G. F. Joyce, Curr. Opin. Struct. Biol. 4, 331 (1994); L.Gold, B. Polisky, O. C. Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 64, 763(1995); J. R. Lorsch and J. W. Szostak, Acc. Chem. Res. 29, 103 (1996);T. Pan, Curr. Opin. Chem. Biol. 1, 17 (1997); Y. Li and R. R. Breaker,Curr. Opin. Struct. Biol. 9, 31.5 (1999); M. Famulok, Curr. Opin.Struct. Biol. 9, 324 (1999).

The length and nucleic acid sequence can be easily modified and therebythe repertoire of possible sensors is almost infinite raging frompreferably about 1 to about 3000 bases. The preferred lengths of thesensing nucleic acid sequences vary from about 10 to about 2000 bases,from about 10 to about 1000 bases, from about 10 to about 500, fromabout 15 to about 400 bases, from about 15 to about 300 bases, fromabout 15 to about 200 bases, from about 15 to about 100 bases, fromabout 15 to about 30, 40, 50, 60, 70, 80, or 90 bases. In a preferredembodiment, the nucleic acids are about 15-24 bases long.

To test the responsiveness of the nucleic acid to an analyte, theisolated or synthesized nucleic acids are labeled with a fluorophore. Asused herein the term “fluorophore” will be understood to refer to bothfluorophores, phosphors and luminophores, and to chromophores thatabsorb but do not emit photons.

Fluorophores provide the means for transducing the interaction of theanalytes with the sensor. For example, fluorophores useful according tothe present invention include, but are not limited to OLIGREEN(Molecular Probes, Inc., Eugene, Oreg.), and other fluorescent dyeslisted in the following Table 2. TABLE 2 Examples of Fluorophores andTheir Excitation and Emission wave-lenghts. Fluorescent Dye Excitation,nm Emission, nm 5-FAM 494 518 Alexa ™ 488 495 520 Alexa ™ 532 531 554Alexa ™ 546 555 570 Alexa ™ 555 555 565 Alexa ™ 568 579 604 Alexa ™ 594590 615 Alexa ™ 647 649 666 Alexa ™ 647 649 666 Alexa ™ 660 663 690Alexa ™ 660 663 690 Allophycocyanin (APC) 650 660 Allophycocyanin (APC)650 660 BODIPY ® 564/570 564 570 BODIPY ® TMR 542 574 BODIPY ® 530/550530 550 BODIPY ® 558/568 558 568 BODIPY ® 630-650 630 650 BODIPY ®630-650 630 650 Calcein 494 517 Calcium Crimson ™ 590 615 CalciumGreen ™ 506 533 Calcium Orange ™ 549 576 C-Phycocyanin 620 648 Cy2 ™ 489506 Cy3.5 ™ 581 596 Cy3 ™ 550 570 Cy5.5 675 694 Cy5.5 675 694 Cy5 ™ 649670 Cy5 ™ 649 670 DiD DilC(5) 644 665 DiD DilC(5) 644 665 dsRed 558 583Ethidium Bromide 518 605 FAM 488 508 FITC 494 518 FluorX ™ 494 519 GFP488 558 GFP Red Shifted 488 507 (rsGFP) JOE 522 555 JOE-514 514 549Magnesium 506 531 Green ™ Magnesium 550 575 Orange ™ Nile Red 549 599Oregon Green ™ 496 524 488 Oregon Green ™ 503 522 500 PBXL-1 545 666PBXL-3 614 662 Phycoerythrin, R & B 565 575 Pyronin Y 555 580 RedReflect 633 633 Red Reflect 633 633 Rhodamine 110 496 520 Rhodamine 123507 529 Rhodamine B 555 580 Rhodamine Green ™ 502 527 Rhodamine 542 565Phalloidin Rhodamine Red ™ 570 590 RiboGreen ™ 500 525 ROX 580 605R-phycocyanin 618 642 R-Phycoerythrin (R- 565 575 PE) SYBR Green 497 520Sypro Ruby 450 610 TAMRA 555 575 Thiadicarbocyanine 651 671Thiadicarbocyanine 651 671 TO-PRO ™-1 514 533 TO-PRO ™-3 642 660TO-PRO ™-3 642 660 YO-PRO ™-1 491 509 YO-PRO ™-3 612 631 YOYO ™-3 612631

Preferred dyes useful according to the present invention includeOLIGREEN or YO-PRO dye (Molecular Probes, Inc., Eugene, Oreg.).

In addition to labeling each oligonucleotide with a single type offluorophore, fluorophore/quencher systems can also be used. Typically,these systems incorporate a fluorophore (e.g., fluorescein) and aquencher (e.g., DABCYL) at the ends of an oligomer sequence that forms ahairpin structure (see, e.g., Tyagi, S. and Kramer, F. R. (1996) NatureBiotech., 14:303-308; Hamaguchi, N., Ellington, A., and Stanton, M.(2001) Anal. Biochem., 294:126-131). In this conformation, the DABCYLquenches the fluorescein fluorescence through fluorescence resonanceenergy transfer (FRET). Upon binding of the oligomer sequence to itstarget ligand, the conformation of the oligomer changes, separating thefluorophore and quencher. This separation decreases the FRET between thefluorophore and quencher, causing a change in fluorescence at thefluorophore emission wavelength.

These energy transfer pairs for fluorophore/quencher systems where boththe donor and acceptor are covalently bound to the same nucleic acid areknown to one skilled in the art. Such energy transfer pairs have beenused to detect changes in oligonucleotide conformation, such as in Tyagiet al. (EP 0 745 690 A2 (1996)) and Pitner et al. (U.S. Pat. No.5,691,145 (1997)). They also have been used to detect cleavage of theoligonucleotide at a point between the donor and acceptor dyes, such asin Han et al. (U.S. Pat. No. 5,763,181 (1998)), Nadeau et al. (U.S. Pat.No. 5,846,726 (1998)), and Wang et al. (ANTIVIRAL CHEMISTRY &CHEMOTHERAPY 8, 303 (1997)). Energy transfer pairs covalently bound tooligonucleotides have also been used to provide a shift in the ultimateemission wavelength upon excitation of the donor dye, such as by Ju(U.S. Pat. No. 5,804,386 (1998)).

Other fluorophore/quencher systems have been described in the art andsuch systems can be used according to the present invention. Forexample, the combination of a non-covalently bound nucleic acid stainwith a covalently attached fluorophore on a single-strandedoligonucleotide hybridization probe has been used to detect specific DNAtarget sequences by monitoring the fluorescence of either the nucleicacid stain or the covalent label, such as described in Lee and Fuerst(PCT Int. Appl. WO 99 28,500). Also, U.S. Pat. No. 6,333,327 disclosesfluorophore/quencher systems for decreasing background fluorescenceduring amplification assays and in ligation assays, and for detectinghybridization.

The nucleic acid can be labeled with the fluorophore at the 3′ region,5′ region of the nucleic acid, or internally. Additionally, theflurophore can be applied dye.

Nucleic acids in the nucleic acid-based sensors of the present inventionare labeled using techniques known to one skilled in the art. Suchmethods include, for example, mixing the nucleic acids with a dye,end-labeling the nucleic acids during oligonucleotide synthesis, orlabeling the nucleic acids during a PCR reaction. According to thepresent invention, any method to attach the fluorophore to the nucleicacid can be used.

Preferred examples of applying or using dyes to label nucleic acidsinclude direct application of dyes, such as for example OLIGREEN andTOTO family of cyanine dimer dyes (Molecular Probes, Inc.), onto thenucleic acids to produce a labeled nucleic acid.

Nucleic acids can also be labeled during their synthesis. Reagents arereadily available (e.g., Glen Research, Sterling, Va.) for addingfluorescent dye molecules to the 3′ and 5′ ends, as well as labeled dTfor inserting the dye molecule within the nucleic acid sequence. Use ofdirect labeling allows control over the precise amount and location ofthe fluorophore within the nucleic acid sequence. Also, a fluorophoremay be added at different locations or multiple fluorophores at severallocations in the nucleic acid sequence which allows development of evengreater variety of sensors.

The sequence and/or structure of the nucleic acid used to construct asensor effects the response profile of the sensor. In preparing thenucleic acid-based sensors, the effect of sequence (and, hence,structure) on the response properties of nucleic acid-based sensors istested. For each sequence tested, the folding structure(s) and meltingtemperature(s) are estimated to determine the effect of a specific DNAstructure on the analyte responses.

The amount of nucleic acids used in producing the nucleic acid-basedsensor effects the response of the sensor to an analyte. For example,effects of DNA quantity were seen in preliminary experiments on thenucleic acid based sensors (FIG. 11). Therefore, for each sensorconfiguration, a range of nucleic acid and dye concentrations (forapplied dyes) are tested for the amount that produces the desiredresult, i.e. a clearly noticeable response to a test analyte.

It is desirable to apply the nucleic acid/fluorophore solution to thesubstrate as evenly as possible. For example, an inkjet applicationsystem can be used. With this system, a piezo-electric inkjet ejects 50nl droplets of solution, which are applied to the substrate in preciselocations using an XYZ positioning system. The inkjet system can be usedto apply nucleic acid/fluorophore solutions to the sensor substrates.

The substrate used to make the sensor can be fabricated of differentmaterials, such as, for example, papers, fiberglass, fabrics made ofsynthetic materials. However, for the purpose of screening/testingnucleic acid/fluorophore combinations for their responsiveness to testanalytes, a glass substrate can be used. The nucleic acid/fluorophorecombination should then be tested on the substrate that is intended tobe used in the sensor to ensure responsiveness will not be effected.

Long-term stability of the nucleic acid/fluorophore -based sensorresponses is important for their use in the present invention.Fluorescent dyes can photobleach upon repeated exposure to excitationlight, and different dyes photobleach at different rates. The presentinvention is designed to minimize photobleaching (by limiting lightexposure to brief 1 msec pulses), and the analyte recognition algorithmsare resistant to changes in signal amplitude. Reducing any possiblephotobleaching, however, will increase the life expectancy of thesensor.

Dried nucleic acids are stable for long periods of time which makes itan ideal sensor material. However, it is possible that the nucleic acidused in the analyte sensors degrades over time thereby altering analyteresponse. The degradation is likely to be minimal and can be easilytested. Analyte responses over repeated sniffs are compared to the datafrom, for example the photobleaching tests described above. Any signaldecrease that cannot be accounted for by photobleaching will suggest anucleic acid degradation effect. If evidence of nucleic acid degradationis found, nucleotide modifications that reduce nuclease degradation canbe used to reduce degradation as described, e.g., for applications toaptamers (see Jayasena, S. D. (1999). Clin. Chem., 45:1628-1650).

The present invention also provides a system for identifying andselecting nucleic acid-fluorophore combinations for their capacity torespond to odors and/or vapor phase analytes. The method includesexposing the nucleic acids-fluorophore complexes to an analyte in avapor phase and comparing the emission of light from the complex beforeaddition to the analyte and during or after exposure to the analyte,wherein difference in the emitted light from the fluorophore between thebefore and during or after exposure to the analyte indicates that thenucleic acid-fluorophore is capable of responding to the analyte.

For example, a plurality of different nucleic acid-fluorophore complexescan be applied onto a substrate, such as a glass coverslip, thesubstrate is then placed in the array scanner and scanned to produce the“before” image. While the chamber is still in the scanner, an analyte invapor phase is injected into it using, for example a syringe. Forexample, a syringe can be used to withdraw some of the headspace vapor.from a container containing a sample analyte. The amount of analyte canvary depending on the size of the chamber and can be as little as about0.25-0.5 ml or about 1, 2, 3, 5, 10, 15 or up to 100 ml. In thepreferred embodiment, about 0.5-2.5 ml of vapor analyte is added to thetest chamber.

The exposure time may be varied from about 1 second to up to 5, 10, 15,20, 25, 30, 40 and 50 seconds and further up to several minutes, forexample, 1, 2, 3, 4, 5 and up to 10 minutes. Most preferably, theexposure time varies between about 25 seconds to about 3 minutes. Afterinjection of the test analyte odor to the chamber with the substrate,the chamber is scanned again to get the “after” image. If there is adifference between the before and after images, in any of the particularcoordinates with a nucleic acid-fluorophore complex, the complex isconsidered reactive to that particular test analyte in vapor phase. Thedifference between the before and after image may be any detectabledifference in the intensity of emission light between the before andafter image. FIG. 6 shows an example of a screen for nucleic acids in amicroarray form, wherein difference of light emission pattern canclearly be appreciated.

The exposure time and test analyte vapor amount may vary because thegoal of using the chamber and scanner is to find any and all nucleicacid-fluorophore spots that respond to the test analytes at all. Themain motivations for the times and volumes is to make sure all spots arecovered by the analyte vapor. In one embodiment, a 2 ml chamber is usedand about volumes of 2-10 rnl of vapor analyte can be injected into thechamber. With high concentration vapor (i.e., saturated vapor), smallervolumes may be sufficient. With low concentrations, higher volumes areneeded to make sure the air in the chamber is sufficiently exchanged.

The gas or vapor phase analyte is preferably injected into the chamberrelatively slowly. For example, for the about 2 ml chamber, iheinjection speed is most preferably about 0.5-1 ml/sec.

The present invention further provides nucleic acid/fluorophore-basedarray sensor element compositions disposed on substrates which may beeither inert or active during analyte sampling and detection. Whileinert supports are typically used in conventional sensing devices, thepresent invention provides for active dye support materials that enhancesensor responses to specific analytes by their unique chemical;physical, adsorption, or optical characteristics. Different substratesupport materials may be employed within a single array where specificsupport materials are matched to specific fluorophores, fluorophorecompounds and nucleic acid/fluorophore mixtures to produce enhancedsensor responses to specific volatile analytes or odors.

Fibrous substrate supports, which enhance sensor response signals for avariety of fluorophores and nucleic acid/fluorophore mixtures arepreferred substrate materials.

An additional advantageous feature of the present invention is inproviding for removable or interchangeable nucleic acid/fluorophore-based arrays, array substrates, or substrate supports to facilitatechanging sensor arrays to match specific analyte sampling and detectionrequirements. In one embodiment, multiple layers of array substrates maybe employed for diversification and enhancement of sensor detectioncapabilities for identifying both broad and specific classes ofanalytes.

One skilled in the art would recognize that it is generally preferred toposition sensor substrates at the appropriate viewing angle and distancefrom light emitting diode excitation light sources and photodiodedetectors so as to provide for optimum sensor signal generation anddetection. In one preferred embodiment, a separate substrate holder maybe provided for positioning and securing array substrates., In analternative preferred embodiment, the sample chamber housing may beconfigured for proper positioning and securing array substrate.

As will be appreciated by those in the art, the. number of possiblesubstrate materials is very large. Possible substrate materials include,but are not limited to, silk, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, teflons, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, and a variety ofother polymers.

In preferred embodiments, optically transparent substrates are employedto permit placement of the substrate between LED light sources andphotodiode detectors as shown in FIG. 10. In alternative embodiments,where the LEDs and photodiodes are placed on the same side of thesubstrate, optically opaque or optically absorbing, reflective, andscattering materials may be employed.

Where conventional flat, planar, curved or non-planar solid sensorsubstrates are used, these substrates are generally self-supporting andsubstrate supports are not required but may be optionally employed.

While conventional flat, planar, or curved non-planar solid sensorsubstrates may be employed, increased sensor surface area can arise fromdepositing dyes on highly convoluted surfaces that include fine fibroushairs of different materials, particulates, porous substrates, or filmsand substrates suspended within the sampling stream. With the innovativesubstrates of the present invention, these preferred substrateembodiments provide enhanced contact and interaction between sampletarget analytes and sensor elements, increased optical response signalper unit of sensor geometrical surface area, and increased opticalresponse signal per unit of sensor volume.

In preferred embodiments, highly permeable, high surface area, textured,fibrous or particulate substrates which have substantial open porosityfor unimpeded transport of vapors and fluids are desired. In preferredembodiments, single or multiply layers of papers, felts, laid, or wovenfibrous materials or fabrics are employed. In alternative embodiments,loosely packed individual fibrous or particulate materials may beemployed.

In a most preferred embodiment, fibrous substrate materials are employedfor signal enhancement. Important considerations in selecting fibroussubstrates are substrate permeability to vapors, high accessible surfacearea per unit volume, response signal enhancement for specific analytes,how the substrate interacts with the sample flow to provide open accessof its external and internal surfaces to analytes for interaction withthe sensing material. While particularly useful fiber substrates areporous, lightweight paper or tissue products, for example Kimwipe™(Kimberly-Clark Corp., Roswell, Ga.), lens papers, facial tissues, andproducts made from cotton, rayon, glass, and nitrocellulose fibers,other fibrous materials employing natural or synthetic fibers such asfelt, batting, textiles, woven fabrics, yarns, threads, string, rope,papers, and laminates or composites of such materials would be equallysuitable as long as they possess the requisite fluid permeability,surface area, surface area to volume ratio, and open porosity for freetransport of vapor and fluid analytes.

Particularly useful inorganic fibers and fibrous material compositionsare natural and synthetic fibers made from glass, ceramic, metal,quartz, silica, silicon, silicate, silicide, silicon carbide, siliconnitride, alumina, aluminate, aluminide, carbon, graphite, boron, borate,boride, and boron nitride. Particularly useful natural or syntheticfibers and fibrous material compositions are polymer fibers made fromaromatic polyamides, nylons, polyarylonitrile, polyesters, olefins,acrylics, cellulose, acetates, anidex, aramids, azlon, alatoesters,lyocell, spandex, melamines, modacrylic, nitrile, polybenzinidazole,polyproplylene, rayons, lyorell, sarans, vinyon, triacetate, vinyl,rayon, carbon pitch, epoxies, silicones, sol gels,polyphenylene-benzobis-ozazole, polyphenylene sulfides,polytetrafluoroethylene, teflon, and low density or high densitypolyethylene. In one preferred embodiment, fiber materials that arehighly absorbent and have good dye retention characteristics, forexample the cellulosic fiber known as Lyorell, may be employed.

In alternative embodiments, fibers may be coated with either chemicalsizing, polymer, ceramic or metallic materials. Chemical sizing such asmodified polyvinyl acetates, organosilanes, coupling agents, anti-staticagents and lubricants may be employed as appropriate.

With respect to signal enhancing sensor substrate properties of thepresent invention, one skilled in the art would generally recognize andunderstand the intended meaning of the term “textured” referring tomaterial surfaces which typically have a distribution of surfacetopographical features, such as high points (peaks) and low points(valleys), ranging from about 100 nm to about 1000 μm RMS. The term“high permeability” generally refers to materials and materialstructures with a high open porosity that provide essentially free,unimpeded access and convective or diffusive transport to, low viscosityfluids, the term “high surface area” generally referring to materialsthat have a surface area of at least 1 M²/g and typically refers tosurface areas ranging between 2 to 500 M²/g. The term “high surface areato volume” generally refers to materials having a surface area to volumeratio of at least 1M²/cm³, and typically refers to surface area tovolume rations ranging between 2 to 1000 1M²/cm³. The terms “porous” or“porosity” generally referring to materials having a distribution ofpore sizes ranging from 100 nm to 1000 μm, and the term “high openporosity” generally referring to materials whose pore distributionssubstantially comprise open pores.

In alternative embodiments, the sensor substrates of the presentinvention may be chemically or physically modified to enhance surfacearea, absorption, adhesion, hydrophobicity, hydrophilicity, repulsion,discrimination or specificity. In some embodiments, the substrate may bechemically altered to provide chemical functionality for interactionwith analytes, such as providing for enhanced affinity, enhancedrepulsion, or steric impediments to analyte adsorption.

In a preferred embodiment, the sensors are made on a substrate ofacid-washed silkscreen, preferably 16xx and sized about 10 mm×12 mm. Thenucleic acid/fluorophore mixture is pipetted onto a silkscreen,preferably about 5-50 μl of nucleic acid/fluorophore mixture is used,and allowed to air dry for about 10-60 minutes, preferably about 20-30minutes, most preferably about 25 minutes. Each sensor is rinsed in 70%ethanol for about 5 minutes, allowed to air dry, then attached tosupports on, for example, glass coverslips.

The nucleic acid/fluorophore-based sensor and sensing system of thepresent invention provides for a rapidly responding, relativelyinexpensive, dynamically configurable, intelligent, portable samplingdevice.

One preferred detection devise useful with the nucleic acid-fluorophore.sensors of the present system is described in detail in the issued U.S.Pat. No. 6,649,416, which is herein incorporated by reference in itsentirety.

The device delivers analytes (odors) in a controlled, pulsatile manner(sniff) to nucleic acid/fluorophore-based sensor array and detectorarray system that generates signals, for example, analog electricalsignals. The number of sensors, detectors, and sampling time points canbe made larger or smaller depending on the classes of analytes that arebeing targeted for detection. Analog signals, for example, are amplifiedand filtered by a pre-amplifier/amplifier module and digitized by ananalog/digital conversion module for storage in a computer memorymodule. All attributes of the sensing process, including odor delivery,sampling, analysis, detection and identification are under programmablesoftware control via a computer.

The sensing device housing the nucleic acid/fluorophore-based array iseasily trained to recognize specific analytes. Training consists ofdelivering a known set of analytes, for example DNT and othernitroaromatic compounds for detection of explosives, to the device, oneanalyte at a time, and storing matrices of values that arespatio-temporal signatures of each analyte in memory. When an unknownanalyte is to be sampled after training, it is delivered to the deviceand a matrix of values acquired from the unknown is compared to matrixtemplates for the variety of analytes stored in memory during thetraining phase. The best match between the unknown and the library ofstored matrices is then determined using a number of differentalgorithms. In one embodiment, the algorithm looks for the best matchafter calculating the sum of the squared differences between each pointin the stored and unknown matrices. In a preferred embodiment, therising phase of each sensor signal is fit by an exponential functioncontaining two parameters describing the signal amplitude and rate ofchange. A matrix of these parameters is then used to represent thesensor array response, and matches are calculated as above using sum ofsquared differences.

The sensing system provides output results in a variety formatsincluding, but not limited to screen displays, plots, printouts,database files, and recorded or synthesized voice messages.

The sensing device of the present invention comprises a sampling chamberhousing an analyte delivery system and a multi-channel array comprisinglight emitting diodes (LEDs) focused through an array of excitationfilters onto individual sensor elements of a sensor array. An array ofphotodiodes, filtered with an array of emission filters, detects emittedlight energy produced by illuminating the sensor elements with LEDexcitation light during interaction with analytes that are drawn intothe sample chamber by the analyte delivery system. The ambienttemperature, humidity, and particulate levels in the sample chamber maybe controlled for improved reproducibility in sampling under a varietyof environmental conditions.

The sensing device generally provides the basic function comprisinganalyte delivery and control (i.e. manipulation of spatial and temporaldistributions; control over temperature, humidity, and duty cycle),detection by a sensor array and transduction of sensor signals into amanipulatable format, analysis of transduction output events, anddynamic feedback control over analyte delivery, detection and analysisfor intelligent sampling and detection and optimization of nucleicacid/fluorophore-based sensor sensitivity and analyte discrimination.

FIG. 9 provides a schematic block diagram showing the general modulardesign aid configuration of the preferred nucleic acid/fluorophore-based sensor array and sensing system components. A detailed schematicof an exemplary sensor array configuration showing LEDs, excitationfilters, sensor elements, (nucleic. acid/fluorophore) sensor arraysubstrates, emission filters, and photodiodes is provided in FIG. 1B.

In a preferred embodiment, the analyte delivery system provides feedbackcontrol over sample temperature, humidity, flow-rate, and the rise andfall times, duration, and frequency of analyte delivery.

Generally, the sensing chamber includes: a) a means for controllingtemperature, humidity, air flowrate, rise and fall times and frequencyof the applied vapor pulses; b) a means for controlling the surfaceproperties of the sensing and non-sensing areas of the chamber (liquid,mucus, or gel lining) in order to impart chromatographic surfaces to thesensing area and/or humidify, dehumidify, or distribute the analyte tothe sensory surface, or to optimize response of the sensing chemistry;c) a means for aerodynamic control over chamber shape which may eitherbe held constant for the duration of analyte delivery or modulated byfeedback control during analyte delivery; and d) a means for active,dynamic feedback control over shape, duration, air flowrate, temporalenvelope, and frequency of analyte sampling (sniffmg). Such feedback maybe derived from examining the spatio-temporal response patterns from thesensor array produced by prior analyte sampling.

The sensing chamber can be optimized for its aerodynamic properties byplacing the detectors in cavities of various shapes. In one embodiment,the sensors may be placed at a bend in the flow path. In an alternativeembodiment, the sensors may be located on the side of the straight flowpath.

The present invention provides the sensing elements that are composed ofnucleic acid/fluorophore mixtures applied to removable sensorsubstrates. In one embodiment, thin films of nucleic acid/fluorophoremixture are deposited on a flat silk, plastic or glass substrate. Inpreferred embodiments, a nucleic acid/fluorophore mixture is depositeddirectly onto fibrous support made from silk, natural or syntheticcellulose, polymers, glasses, ceramics, metallic, or other materialsusing an ink jet printer. The use of fibrous dye substrates dramaticallyincreases the magnitude of the response signals, which improves analytedetection and discrimination of the device. In an alternativeembodiment, thin nucleic acid/fluorophore films can be suspended freelyacross a perforated removable solid support which is placed in thecenter of the air flow stream, thereby exposing both sides of thenucleic acid sensor to volatile compound analyte.

The sensing device according to the present invention usesinterchangeable, removable sensors or sensor elements comprising asupport wherein nucleic acid/fluorophore complexes are attached. Easilyremovable sensors facilitate rapidly changing sensing sites forimproving the sensitivity and optimizing discrimination for specificanalytes in a variety of sampling applications. This feature furtherprovides for rapid screening of different nucleic acid/fluorophoremixtures for evaluating new nucleic acid sequences and or structures ordifferent fluorophores for use in sensors and also for evaluatinganalytical detection algorithms.

The size, thickness and surface area of sensor element sites may bemodified to optimize sensitivity and discrimination and to efficientlycouple sensor elements to light sources and detectors. Generally, alarger sensor geometric area and a close matching of the sensor elementgeometric area with photodetector area will provide better sensitivity.

The cross-reactive sensor array of the present invention may compriseeither narrow or broadly responsive sensor elements. The number ofsensor array elements can be configured for specific samplingapplication requirements. Specific sensors for defined analytical taskscan be chosen from among the many possible sensing element sites presentin the array. Sensor and array configurations may be modified throughthe addition of LED-sensor-photodiode-filter channels depending on therequirements of a particular analyte discrimination task.

In one preferred embodiment, multiple sensor arrays and array substratesmay be deployed in the sampling chamber. Such multiple arrays maycomprise a series of hierarchically organized sensor arrays such thatthe first interaction and sampling of the analyte is with a broadlyresponsive sensor array and, subsequently, the analyte sample isautomatically diverted for additional sniffs, on the basis of analyticalinformation fed back from the computer, to specific second order arraysdesigned to detect and identify the specific type of analyte. Thus, aplurality of sensing arrays may be arranged hierarchically so that everfiner discriminations can take place successively along the pathway.Additionally, the longevity of sensors can be extended by redundantarrays that are protected from exposure until needed, by delivery ofanalytes as short pulses, and by reducing light exposure by rapidlypulsing LEDs. To further reduce light exposure, low light excitationlevels can be used if high sensitivity photodetectors such as avalanchephotodiodes are employed. Rapid short pulsing of analytes preventssensing surfaces from saturating, thereby improving sensor recoveryfollowing analyte exposure.

For enhanced, smart mode operation, the number of array sensors used insampling or detecting an analyte may be modified, in real-time duringeither actual sampling or post-sampling data analysis using “on-the-fly”intelligent feedback control. By way of example, if a specific sensor isunresponsive to a particular. analyte sample, the corresponding sensingchannel may be automatically removed from consideration by a smartsampling or analysis algorithm which provides feedback control to themicrocontroller. In addition, the weighting of individual sensors in theanalysis and detection algorithm may be adjusted based on the signalcontribution of individual sensors. Given that individual sensors havedifferent breadths and peaks of response, sensor weighting will vary fordifferent analytes.

In one preferred embodiment a 16 or 32 channel sensor array is employed.For example, it is anticipated that an optimized array of thirty-twosensor elements should have the capability of detecting anddiscriminating at least 1000 different analyte types. Because thenucleic acid/fluorophore-based sensor materials employed provide almostinfinite diversity in their variety and therefore their analytedetection capability and can be selected to have appropriately broadspectra of response, different optimized sensor arrays can be selectedfor particular analyte detection tasks.

Typically, epi-illuminating optics are employed in conventionalfluorescence sensing systems. Epi-illuminating optics require relativelycomplex dichroic mirror arrangements for each channel where a differentexcitation and emission wavelength is used. Thus, in theepi-illumination format an excitation filter, a dichroic mirror, and anemission filter are required for each wavelength. The sensing system ofthe present invention employs a trans-illumination configuration whereonly excitation and emission filters are needed. Since theepi-illumination mode typically requires critical optical componentalignment and is sensitive to vibration and movement, thetrans-illumination mode of the present invention is advantageous forrobust, compact, portable sensing devices for field sampling of ambientenvironments.

A schematic diagram of the optical detection system of the presentinvention is provided in the block diagram of FIG. 9. FIG. 1B provides across-sectional view of the sampling chamber that schematically showsthe configuration and relative orientation of individualLED-photodiodes-optical filters-sensor sets within the sampling chamberhousing. For simplicity, the cross-sectional view in FIG. 1B shows onlytwo sensing channels, comprising two LED-photodiode-filter-sensorchannel pairings. FIG. 1A shows a view of a sixteen sensor arrayconfiguration. It is important to note that the partial arrayconfigurations shown in FIGS. 1A and 1B are merely used to demonstrate,by way of example, the relative orientation and positioning of thesensors, filters, photodiodes and LEDs in the sampling chamber and arenot intended to indicate any limitation in the size of sensor arraysthat may be employed in the present invention. The actual sensing deviceof the present invention may employ larger or smaller arrays and anynumber of sensing channels with correspondingLED-photodiode-filter-sensor sets. For example, in one preferredembodiment, 32 LED-photodiode-optical filters-sensor channel sets areemployed. The number of sensor array channels may be increased ordecreased depending on specific sampling applications and analytediscrimination requirements.

An example of the configuration and relative orientation of LEDs,photodiodes, excitation filters and emission filters, sensors and sensorarray substrate is shown schematically in FIG. 10. While an eightsensor-LED-photodiode-filter module is shown in FIG. 10 by way ofexample, larger and smaller modules and arrays may be constructed basedon specific sampling and detection needs. For example, in oneembodiment, a 32 element sensor array may be assembled from four modulesaligned side-by-side with eight sensors in each module. As shown in FIG.10, a plurality of LEDs are mounted on black plastic support by drillingtwo columns of four 3 mm holes in a 2×4 array configuration. The LEDsare press fit into the mounting holes and may be readily removed forreplacement. A photodiode support with the same dimensions is used formounting a plurality of eight photodiodes in a 2×4 array configuration.Both the LED and photodiode arrays are mounted in columns with pair rowspacing of about 6 mm center to center and interpair spacings of 8 mmcenter to center. Column spacing for both the LED array and photodiodearray is 15 mm center to center.

As shown in FIG. 10, 12.5 mm diameter excitation filters are mounted onan approximately 30 mm×30 mm×6 mm excitation filter support formed bydrilling four 12.5 mm holes in a black plastic support plate toaccommodate the filters in a 2×2 array configuration. Other filterassembly configurations, containing a larger or smaller filter arraywith larger or smaller filters may be employed in other embodiments. Asimilar emission filter support with the same dimensions as theexcitation filter support is fabricated for mounting four emissionfilters. The emission filters and excitation filters are mounted totheir respective supports with conventional set screws. The resultingexcitation filter support assembly is attached directly to the frontface of the LED support assembly and the emission filter supportassembly is attached directly to the front face of the photodiodesupport assembly with conventional mounting screws.

A plurality of nucleic acid-based sensor elements are applied eitherdirectly to a transparent sensor array substrate, for example a glasscoverslip, as coatings or droplets. Alternatively, where porous orfibrous sensing elements are employed, these may be attached, forexample, taped, glued, or clamped, to a transparent sensor arraysubstrate, or suspended over openings or perforations in an arraysupport which may be either transparent or opaque. Removable,interchangeable sensor array substrates, or array support substrates,can be mounted flush with the front face of the emission filter supportusing a substrate support holder. The substrate support holder is formedby attaching, for example by gluing, a shaped, preferably U-shapedsubstrate support frame and a shaped substrate support facing to thefront fact of the emission filter support. The sensor array substrates,or array support substrates, are, for example mounted in a slot orchannel formed by the substrate support frame, support facing and frontface of the filter support. The substrate support assembly provides forrapid removal and replacement of the interchangeable array substrates orarray support substrates.

The sensor array may comprise either a single sensor array module, asshown in FIG. 10, or a plurality of sensor modules aligned edge-to-edgeto form a multi-module array containing a large number of sensorelements. The bottom edge of both the LED-excitation filter modulesupport assembly and the photodiode-emission filter-sensor modulesupport assembly are secured to a chamber support plate withconventional mounting screws. In this configuration, the excitationfilter side of the LED assembly faces the sensor array side of thephotodiode assembly. The LED and photodiode modules, or plurality ofmodules, are preferably aligned parallel to one another with spacingbetween the two modules adjusted to optimize illumination of the sensorarray elements by the LED array. In one preferred embodiment shown inFIG. 10, this spacing is approximately 5 mm. In one preferredembodiment, a 32 sensor array is formed by mounting four eight sensormodules to the chamber support plate. Other configurations using largeror smaller sensor modules and a fewer or greater number of modules maybe employed to accommodate smaller or larger arrays by adjusting thesize of the LED, photodiode, filter and sensor supports and chambersupport plate and adjusting the spacing between opposing LED andphotodiode modules to optimize illumination of sensor array elements bythe LED array.

Commercially available, optical bandpass excitation filters for LEDlight sources and emission filters for photodiode detectors wereobtained from Andover Corp. (Salem, N.H.) and Coherent Inc. (SantaClara, Calif.). While these filters are available in ¼ to 1½ inch sizes,½ inch filters were used in the preferred embodiment. By way of example,FIG. 10 shows schematically the relative orientation, configuration andspacing of excitation and emission filters for an embodiment whichemploys 32 sensors and sensing channels. For simplicity, FIG. 10 showsonly one of four eight-sensor modules employed in a 32 channel sensorarray. In this embodiment, with four sensor modules, 16 excitationfilters are arranged in a 2×8 array with a center to center distance of15 mm. With this embodiment, each emission filter covers a pair of twoadjacent photodiodes having a 6 mm center to center spacing. In thisparticular embodiment, the 32 sensor elements in the array were alignedwith the center of the LED-photodiode pair sight line. Other embodimentsare envisioned where each sensor channel has its own individualexcitation and emission filter or where more than two sensor channelsshare each excitation and emission filter. For example, for YO-PRO andOligreen dyes, an excitation filter of 450 nm with a 40 nm bandwidth,and emission filters with 550 nm with a 70 nm bandwidth can be used(Coherent Inc., Santa Clara, Calif.). Dyes such as BOBO-3 and Cy3(tm)require longer wave lengths which one skilled in the art is capable ofselecting.

Illumination of sensor elements with excitation light energy may beaccomplished with any appropriate light source. Thus, filtered lightemitting diodes (LEDs), solid-state lasers, or incandescent lightsources of the appropriate wavelengths for the dye indicators being usedmay be employed. In a preferred embodiment, each LED light is passedthrough an excitation filter matched to a specific sensor element dyeexcitation wavelength. Where excitation filters are employed, broad-band(“white”) LEDs with appropriate wavelength filters may be used.

Unlike other sensors, by providing individually filtered sensingchannels, the present invention enables simultaneous sampling atmultiple excitation wavelengths and multiple emission wavelengths withdifferent sensor elements. The. present invention uniquely provides forindividual control over the amplitude, duration, and duty cycle ofillumination for each sensing channel in the array. Control over noiseis exerted by feedback. Control over response to ambient light andoptimization of signal detection, including reduction of fluorescent dyebleaching, is accomplished by switching and modulating LED output andcoordinate amplifier detection at various frequencies, ranging fromkilohertz to megahertz. Control over ambient light interference may beachieved by phase locked LED flashing and photodiode detection.

In the present invention, nucleic acid-based sensor elements areilluminated directly by focused, light emitting diodes (LEDs) of thecorrect wavelength for each sensor dye material. Other advantagesachieved from using LED excitation light sources are low powerrequirements, cooler operating temperatures, and high light output oversmall area. Additionally, by employing LED light sources for each sensorchannel, each LED channel can be rapidly and independently switchedelectrically without use of a mechanical shutter. The LED channels canbe individually modulated electrically at high rates by feedback fromthe microcontroller. In addition, the LED channels can be individuallyfiltered for presenting different excitation wavelengths in parallel,thereby avoiding serially and mechanically switching filters duringarray measurements.

Examples of LEDs useful according to the present invention for thenucleic acid based sensors include, but are not limited to Hosfelt#25-365, Ultra Bright Blue LED, rated at about 466 nm. Other LEDs usefulaccording to the present invention can be selected according towavelengths appropriate for each and every fluorescent molecule that canbe attached to the nucleic acids as shown in the Table above.

The LED's are turned on and off under computer control. Since thesedevices can respond at high speeds, up to megahertz frequencies, theyare typically flashed at kilohertz frequencies in order to reducebleaching. Such switching speeds cannot be achieved using mechanicalshutters. The rapid switching capacities of LED's are utilized to flashthem on and off in order to reduce sensor bleaching during dataacquisition, thereby reducing total light exposure by shortened dutycycle during sample sniffs. LEDs are rapidly flickered so that light isonly on during the time when data are being taken and then turned offbetween data points and between trials.

While a variety of photodetectors such as photomultiplier tubes (PMTs),charge-coupled display device (CCD) detectors, photovoltaic devices,phototransistors, and photodiodes may be used for detecting sensorresponse signals, in a preferred embodiment, filtered photodiodedetectors are employed. In another preferred embodiment, highlysensitive avalanche photodiodes may be employed. Photodiode detectorshave distinct advantages compared to conventional CCD camera detectorssince they enable independent control and modulation of individualchannel optical filtering, current/voltage conversion, signalamplification, and temporal filtering. Other specific advantages are lowpower consumption, relatively simple electronic circuitry, highsensitivity, configurability, multiple array formats (e.g. circular,square, or linear arrays), fast high frequency response at megahertzfrequencies, low noise, wide dynamic range, and use with low frequencycircuits.

In the nucleic acid-based sensing device of the present invention, anarray of filtered photodiodes is employed where each filtered photodiodeis either aligned with one filtered LED or, altematively, groups offiltered photodiodes may be illuminated by a single filtered LED. Theindividual photodiodes are each aligned with an individual sensorelement site with an optical emission filter that is appropriate for thespecific dye employed by the individual sensor. Different emissionfilters may be used for each photodiode or, alternatively, one emissionfilter may be shared by multiple photodiodes. Photodiode signal noise iscontrolled by feedback. Additionally, feedback control is exerted overthe signal sampling duration and time course. Differential signal inputsmay be employed with a separate control sensor and individual samplingsensors. In one preferred embodiment, highly sensitive avalanchephotodiodes may be used to permit lower required LED intensity forsensor excitation thereby reducing sensor photobleaching.

In one embodiment commercially available EG&G VTP 1232 photodiodes(EG&G, Inc, Gaithersburg, Md.) and 12.5 mm emission filters (AndoverCorp., Salem, N.H. and Coherent Inc., Santa Clara, Calif.) were used. Ina preferred embodiment, large area photodiodes (Hamamatsu part no.S2387-66R) are used. Specific emission filters used in conjunction withthe photodiode detectors are discussed above.

While sensors may share the same LED, photodiode and excitation/emissionfilters, in alternative embodiments, separate LED, photodiode, sensor,and excitation/emission filters may be employed for each of sensorelement and sensing channel. In one embodiment, individual sensorelements and sensing channels may employ different sensing materials,different excitation wavelengths, and/or different emission wavelengthssimultaneously. One skilled in the art may increase or decrease both thesize of the sensor array and number of sensing channels, following theteachings disclosed herein.

In one embodiment, all LEDs are powered by a single constant voltagecircuit. The changes in fluorescence as a result of the odor interactingwith the sensing material is detected by a photodiode and current tovoltage (I/V) converter originally designed by Warner Instruments(Hamden, Conn.) and now commercially available from Red Shirt ImagingInc. (Fairfield, Conn.). There is one I/V converter and amplifier/filterfor each detector channel. The unique feature of thisconverter/amplifier configuration is that when the LEDs are activatedprior to sample delivery, the background fluorescence signal produced bythe sensor elements may be offset by resetting the amplifiers to abaseline value so that a full range of high gain amplification may beused to observe small changes in the signals generated by analytesduring sampling. In addition, the amplifier board has the option forsoftware control to be exerted over the gain and the filter timeconstants for all the channels. Photodiode output is digitized using a12 bit A/D converter. In a preferred embodiment, each LED is poweredindependently by its own constant current circuitry. The output currentof each photodiode is converted to voltage and digitized to 20 bitsusing an integrating preamp/AD converter IC manufactured by Burr-Brown(DDC112). The DDC112's provide separate gain control for each sensorchannel. Circuitry containing two programmable logic devices (PLD;Xilinx part no. XC95108-15PC84C) generates the high speed timing controlsignals for the 16 DDC112 chips.

Thus, in addition to being able to manipulate the onset and duration ofthe illumination and of the sniff as described above, the time constantsand gain of the amplifiers can also be controlled in real time duringdata acquisition. These hardware features offer distinct advantages foroptimizing the response of the sensing device for detection,discrimination and identification of analytes or odors of interest.

Generally, the nucleic acid/fluorophore-based sensing system of thepresent invention analyzes spatial-temporal patterns of data output fromnucleic acid-based sensor arrays in order to characterize and identifythe delivered sample or its analyte components. Useable information fromthe sensing array is obtained from the pattern of sensor responseactivity generated by all sensor elements over time and is evaluatedusing statistical measures such as information theory. Patternrecognition algorithms including template comparison, neural networks,principal components analysis, etc. may be implemented either inconventional digital CPUs, in neuronal network simulator chips, or inanalogue neuronal network computers. Additionally, algorithms based onbiologically based neuronal connections from the olfactory system andother neuronal circuits in the brain may be employed.

The analytical circuits of the present sensing device provide therequisite hardware support for the detection, discrimination andidentification capability of the sensing system.

The present invention uses temporal control over stimulus presentationand the examination of the resulting changes in sensor output over time.Unlike other designs, with the present invention analyte presentation tothe sensing sites is carried out by negative pressure ‘sniffing’, ratherthan by positive pressure pulsing which requires samples to be enclosedin confined containers. Additionally, the present invention usessniffing parameters that can be electronically modulated by feedbackfrom via computer control and flow rate, sniff duration, and temporalprofile can be adjusted and modulated for specific sampling environmentsand target analytes to detect ambient odors drawn into the sensingchamber. Sampling modulations can be carried out in real time so thatsubsequent sniffs can be modified by the preceding ones. With the smartsampling mode capability of the present invention, a computer turns thesniff on and off and can modulate and control sniff parameters duringsampling.

Target samples of known analytes (odors), either pure compounds orcomplex mixtures, are required for training the sensing device andidentifying unknown analytes in sampled fluids.

For all training runs, initially a clean air test sniff is first takenby initiating the automated sampling sequence which provides for turningon the LEDs, taking digitized data from the photodiodes, measuringbackground fluorescence and storing this in memory, turning on the sniffpump, turning off the pump, terminating data acquisition, and turningoff the LEDs. The device is then trained for target analytes by placingthe target analyte sample container into position and initiating theautomated sampling sequence. The sequence of sampling and dataacquisition events for target analytes is the same as for the airbaseline sample. This training sequence is repeated for each targetanalyte of interest and response data are stored in the microcontrollercomputer RAM memory module.

After analyte presentation and data acquisition using a device, such asa device described in the U.S. Pat. No. 6,649,416, evaluation circuitsand algorithms characterize the spatio-temporal response data of thearray either via pattern recognition algorithms, template matching, aneural network, statistical analysis, or other analytical methods knownto be useful for data analysis from multiple points. Results may bedisplayed on screen, spoken by voice synthesis, or plotted as athree-dimensional response surface of fluorescence changes from eachsensor at each time point during sampling. If sensing device is onrobotic vehicle, results are processed for feedback control and decisionis made to stay on course or execute an appropriate maneuver.

Optionally, where multiple samples or complex mixtures containingmultiple analytes are being sampled, with data sampling and acquisitionmodifications based on intelligent feedback via smart algorithms. Thus,real-time, on-the-fly feedback can dynamically modulate either LED,photodiode, or sniffing hardware settings, or alternatively, analytesampling parameters such as, sample duration, rise time, relaxationtime, delay from previous sniff, amplifler gain and time constants maybe modified. These modifications may be imposed on the next dataacquisition within the same sampling trial until detection andidentification of the analyte occurs.

The software program explicitly controls the pre-bleaching phase, theduration for which the LED's illuminate the sensors, the onset of dataacquisition, the application of the analyte, the duration of analytepresentation, the cessation of analyte application, the duration of theintegration time for each data point, the number of time points, and theinterval between time points. All of these parameters can be modulatedeither by direct operator intervention or, alternatively, by programmingthe microprocessor with smart algorithms that modify the sampling, dataacquisition, or analysis steps through real-time feedback control.

The data are filtered, smoothed, statistically evaluated, compared withlibraries of stored templates for odor identification, and/or operatedon by any of the algorithms discussed below. The data are typicallystored in memory as an array of numbers representing the temporalchanges in fluorescence in each sensing channel.

1. Detection Methods and Algorithms

A. Evaluation of Synchrony, Response Signals and Noise Characteristics

To improve the detection and discrimination capability of the sensor ofthe present invention, additional algorithms may be employed to evaluate“synchrony” of response data across different sensor elements toidentify small response signals and reject noise. Evaluation of“synchrony” refers to analyzing how signals coming from identicalsensors are similar in the context of when they occur during the sniffcycle. The field that encompasses analytical algorithms is very largeand many analytical approaches are available. Due to the features of thepresent invention, such as the use of multiple detector channels withdifferent wavelengths, use of single or multi-pulsed analytepresentation, and the ability to acquire data from sensor elements inparallel rather than serially, the design of the present inventionenables consideration of a number of alternative algorithms beyond thosethat are conventionally used in artificial noses. Additionally, inpreferred embodiments algorithms which are based on biological circuitsmay be employed (see J. White, et al., Biol. Cybern. 78:245-251(1998);J. White, et al., Anal. Chem. 68(13):2191-2202 (1996), whichpublications are incorporated herein by reference in their entirety).The device of the present invention may employ synchronously occurringsignals in some embodiments since sensor response data are acquiredsimultaneously in parallel.

Detection Algorithms

The degree to which the response matrix of a test substance correspondsto one of the target analyte library matrices stored during the sensortraining phase can be evaluated in a number of ways.

In one preferred embodiment, the rising phase of each sensor signal isfit by an exponential function containing two parameters describing thesignal amplitude and rate of change. A matrix of these parameters isthen used to represent the sensor array response. Matches are determinedfrom the sum of the squared differences between each parameter in thetest matrix and the training matrix. The smallest sum is used toidentify the best target analyte match.

In an alternative preferred embodiment, a supervised, for example backpropagation, neural network approach may be employed. Examples of thesemethods are provided in J. White, et al. “Rapid Analyte Recognition In ADevice Based On Optical Sensors And The Olfactory System”, Anal. Chem.68(13):2191-2202 (1996) and S. R. Johnson, et al., “Identification OfMultiple Analytes Using An Optical Sensor Array And Pattern RecognitionNeural Networks”, Anal. Chem. 69(22):4641-4648(1997).

In another preferred embodiment, analytical circuits based on theolfactory system may be employed as disclosed by J. White, et al., “AnOlfactory Neuronal Network For Vapor Recognition In An Artificial Nose”,Biol. Cybern. 78:245-251(1998).

In another preferred embodiment, unsupervised neural networks may beused. Principle component analysis and multidimensional scaling are, ineffect, unsupervised statistical methods for reducing dimensionality.Generally, unsupervised neural networks organize high dimensional inputdata into lower dimensional representations. For example, assuming oneembodiment of the present device with 32 sensors and 20 time points, atotal of 640 data points may be collected. In this embodiment, eachanalyte presentation can thus be thought of as a point in 640-dimensionspace, which, while difficult to visualize, may be mathematicallymanipulated. By averaging across sensors and time, the datadimensionality may be reduced, but typically data dimensionality aboveabout four dimensions is rather difficult to visualize.

Self-organizing maps (SOMs) are unsupervised neural networks that reducedata dimensionality. Such SOM methods are attractive for representingartificial olfactory system data because they give a visualization of“odor space”. In other words, a map of relationships among variousanalytes can be produced during training; then during testing, thelocation of a test analyte on the ‘map’ indicates the relationship ofthe analyte with respect to this ‘space’. Thus, SOMs may help tovisualize relationships among analytes, rather than simply indicatingthe similarity of an unknown analyte to a target. Examples of SOMapproaches which may be particularly useful for analyte detection,discrimination and identification are disclosed by T. Kohonen. et al.,“SOM-PAK: The Self-Organizing Map Program Package”, Report A31, HelsinkiUniversity of Technology, Laboratory of Computer and InformationScience, Espoo, Finland (1996) and T. Kohonen, Self-Organizing Maps,Series in Information Sciences, Vol. 30, 2nd ed., Springer-Verlag,Heidelberg (1997), which publications are incorporated herein by thisreference.

Sampling and Detection Parameter Modulation

Upon evaluation of the response matrices generated by the standards usedfor training, modifications in sniffing parameters, gain settings,and/or filter settings may be made for actual sampling of ambientfluids. In a standard operating mode, these modifications may be madethrough interventions of an operator who manually changes sampling anddata acquisition parameters through the programmable microcontroller orby keyboard entry. In alternative smart operating modes described insubsequent sections, these modifications may be made automatically,on-the-fly by smart sampling and detection algorithms that directmicrocontroller operations.

Whether and how much such modification improve sensing performance maybe evaluated by examining sensor responses after feedback anddetermining, by some pre-determined or analytically-derived criterion,whether current sample data are better or worse than data obtained on aprevious run. Modifications may also consist of differentially weightingthe influence of sensors, so that those sensors that give the bestsignals have a greater impact in the recognition algorithms. This can bedone in a number of ways, such as eliminating sensors that give littleor no signal so as to reduce noise, normalizing the remaining signals tosome standard value in order to use the maximum range available, orchanging the analyte sampling and stimulus acquisition paradigm tooptimize sniff sampling parameters.

“Smart Mode” Operation

One example of an embodiment of the smart mode sampling capability ofthe present invention is where the number and duration of analytesamples taken during a sample session are controlled by way of real-timefeedback and control loops for improving detection, discrimination andidentification of analytes. In other embodiments, alternative smart modeparameters and device sampling configurations may be manually orautomatically selected during training and sampling via device menuoptions. Smart mode sampling configurations may be used alone or in avariety of combinations and permutations. In one anticipated embodiment,an automated training algorithm may be employed to optimize parameterselection and sampling configuration in order to provide the bestdetection and discrimination capability for specific analytes ofinterest. Specific examples of alternative smart mode sampling optionsand parameter configurations are described below.

Sampling Parameters

Sniff Parameters—Sniff Duration

For sensors that respond slowly to a particular analyte, increasing thesniff duration leads to increased signal amplitude and hence improveddetection accuracy.

Sniff Parameters—Number of Sniffs.

In the simplest implementation, signals across multiple sniffs may beaveraged to improve signal-to-noise. However, different sensors exhibitdifferent long-term responses to multiple sniffs (providing eitherincreasing signal, decreasing signal, or constant signal over a seriesof sniffs). Monitoring these changes over sniffs (rather than simplyaveraging the signals) could provide additional information for analytediscrimination.

Sniff Parameters—Sniff Dynamics (Rise Time, Fall Time).

The rate and extent of sample chamber valves opening and closing may becontrolled to modify sampling (sniff) dynamics.

Changing the sniff dynamics may enhance differences in the rising andfalling phases of the sensor response.

Sniff Parameters—Sniff Velocity.

In one anticipated embodiment, a digital-to-analog line may be used tocontrol a transistor that changes the voltage supplied to the sniff fanand thereby alter fan velocity. Changing sniff velocity, in conjunctionwith changes in sniff duration, can provide optimized exposure of thesensors to particular analytes.

Sniff Parameters—Exhalation Velocity.

As with changing sniff velocity, a change in exhalation velocity in anembodiment with two fans would alter the rate at which analyte is purgedfrom the. sensors. In a system with a single fan, the velocity of thatfan between sniffs can be similarly altered. The dynamic sensor responsemay then be monitored in subsequent sniffs for improved analytediscrimination. LED Intensity.

While higher LED intensity leads to more rapid photo-bleaching andsensor degradation, it also tends to yield larger sensor responsesignals during analyte exposure. In one smart mode embodiment, normalsampling would be made at lower LED intensity and, where small responsesignals are present, LED intensity may be increased incrementally untilreliable response signals are produced for analyte detection. This smartmode would tend to extend sensor lifetime by operating at minimum LEDintensity to reduce photobleaching.

LED Wavelength.

The excitation wavelength of the LED may be modulated. LEDs arecommercially available that produce three separate wavelengths. Thewavelength of conventional LEDs may be modulated by changing appliedvoltage and flicker frequency. The capability for changing LEDwavelength may permit the device to optimally excite the sensors and tochange that excitation over sniffs to improve discrimination.

Amplifier Gain Settings.

Under typical sampling conditions, the highest gain settings areemployed. Under such a condition, some analytes produce sensor signalsthat saturate the amplifier. By providing for adjustment of gainsettings during smart mode sampling, if an amplifier channel saturates,an additional sniff at a lower gain setting would provide more accuratetime course and amplitude information.

Amplifier Temporal Filter Settings.

In embodiments incorporating amplifiers containing integral temporalfilters, changing the filter settings may be used to improve thesignal-to-noise characteristics of the individual sensor channels. Dataacquisition and A/D conversion are closely correlated with LED pulsetiming. However, some detection enhancement may be achieved by modifyingthe timing of data acquisition during an LED pulse for improved signaldiscrimination for specific analytes; modulation of this parameter maytherefore improve detection and identification of certain analytes.

Gain and Temporal Filter Settings for Individual Channels.

While one current embodiment of the amplifier electronics allowmanipulation of gain and filter settings globally (i.e. gain and filterchanges apply to all channels simultaneously), in alternative sensorembodiments, individual sensor channels may also be manipulated forsmart mode sampling and detection.

Smart mode training and sampling procedures using these and otherparameter variations are discussed in greater detail below.

Smart Mode Training

Smart mode training can be divided into two sections: first, theparameters defining the “primary” sniff are determined, followed by adetermination of parameters for any “secondary” sniff(s) that may benecessary. The constraints for the two sets of parameters are different:The primary sniffs are applied at regular intervals over long periods oftime and should have minimum impact on sensor lifetime since they exposethe sensors to as little light as possible to reduce photobleaching andto as little analyte as possible to prolong sensor lifetime and shortenrecovery time. Secondary sniffs are intended to generate signals thatproduce better discrimination.

Photobleaching and Bleach Runs

Exposing a fluorescent sensor to prolonged excitation light producesphotobleaching, decreasing the fluorescent output of the sensor. Thisfluorescence recovers over time after the excitation light is turnedoff. In embodiments where sensors are exposed to prolonged excitationlight during acquisition of response data at variable intervals, thereappears to be more variability in sensor response. Preferably, responsedata are acquired at regular 15 second intervals. Sensor bleach runsestablish this regular interval before data are actually acquired. Thebleach runs are repeated until the signals from the sensors stabilize.In preferred embodiments using short excitation light exposures (1-5ms), variability across sniffs due to photobleaching is greatly reduced.

In embodiments using longer excitation light exposure, bleach runs areacquired either with or without sniffing a blank air sample. Theresponse matrices from these runs are compared to the previous run bycalculating the sum of squares (SS) difference for all data points. Forthe first run, the comparison is to a matrix of zeroes. If the SSdifference is stable, where successive SS differences change little,training target sampling is initiated. If the SS difference is unstable,a 15 second inter-run delay time is used and then the bleach run isrepeated. While the operator may evaluate the SS difference stabilityvisually, this process may be automated by setting a criterion whichprovides for minimum changes in successive SS differences; when thatcriterion is reached, the program continues and training target samplingis initiated.

Smart Nose Testing

Smart Nose testing a single analyte can occur in two stages. First, aprimary sniff is taken and, if the primary sniff produces a good matchto a target, that match is reported. Secondly, if the primary sniff doesnot produce a good match, one or more secondary sniff(s), if defined bytraining, are taken. If a match criterion is not reached, the matchingdifficulty is noted and the closest match reported. If the qualitycriterion is reached, the match is reported.

The photodiodes useful according to the present invention are generallymore sensitive than and have larger dynamic range than individual pixelsof conventional CCD camera detectors. The detection surface area ofindividual sensor photodiodes in the present device is larger thanindividual pixel areas of conventional CCD camera detectors.Additionally, due to the surface area of the LEDs and photodiodesemployed in the present invention, larger sensor element areas may beemployed and sampling is conducted over a larger geometric surface areaof individual the sensor elements. Furthermore, the high porosity highsurface area sensor substrates of the present invention, further enhancesensor response signals due to a substantial increase in sensor surfacearea to volume ratios and the volumetric sampling of sensor responsesignals generated within a three-dimensional substrate-sensor volume.

The enhanced sensitivity of the present sensors may be further augmentedby utilizing multiple layers of sensing material ‘suspended’ in the airstream, employing larger surface area sensor elements and larger surfacearea photodiodes, and/or using replicates of multiple identicaldetectors in the sensor array from which signals are combinedelectronically. Replicates of different sensing materials may beincorporated into different sensor channels. Using replicates providesadvantages not only with respect to the duplication of data to verifymeasurement reproducibility, but also with regard to reducingnon-correlated noise from electronic components such as amplifiers.

The invention further provides a method of selecting a nucleic acidcapable of responding to a vapor phase analyte, said method comprising:a) contacting the nucleic acid labeled with a fluorophore with ananalyte in vapor phase; and b) measuring the emission proflile of thefluorophore in the presence and absence of the target analyte, wherein adifference in the emission profile indicates that the nucleic acid isresponsive to the analyte in vapor phase.

The nucleic acids according to the method can be prepared by any methodknown to one skilled in the art including, but not limited tooligonucleotide synthesis using method described earlier, polymerasechain reaction (PCR) using any DNA as a template, or isolating nucleicacids from any source, including but not limited to eukaryotic andprokarytotic cells, nucleic acid libraries in bacteria, cosmids, yeastartificial chromosomes and such.

Nucleic acids may be labeled using any fluorescent label and methodknown to one skilled in the art. In one embodiment, the nucleic acidsare labeled with Cy3(tm) label. A set of nucleic acid oligomers aredesigned, wherein the internal sequence is a random sequence and the N-and C-terminal ends have an essentially same sequence or an anchorsequence. An example of a random oligo nucleotide with random 20-mersequence in between is T(15)CCN(20) AAACATTGCGAAGAAA (SEQ ID NO: 6).Such random primers with fixed anchor ends can then be used to create alibrary by amplifying nucleic acids isolated from any source, such asbacterial DNA. Once the random sequences are amplified, they can becloned into a library, for example a plasmid library, using methodsknown to one skilled in the art. Such libraries can then be amplifiedusing, for example, PCR with a forward primer haveing a sequence T(15)CCin combination with a Cy3(tm) labeled reverse primer T*TTGTAACGCTTCTTT(SEQ ID NO: 7).

In accordance with the present invention, if the nucleic acid isinternally labeled, any position is acceptable. For example, the labelcan be located in or near the 5′-end, or in or near the 3′ end.Additionally, applied dye such as YO-PRO and Oligreen can also be used.

Once the nucleic acid is labeled it is purified. Purification may beperformed using any method known to one skilled in the art. In theexample outlined above, oligo dT spin columns (available, for examplefrom Amersham Biosciences Corp., Piscataway, N.J.).

The microarray slides useful according to the present invention can beproduced using a variety of surface substrates and methods of depositingnucleic acids on the surfaces. For example, glass coverslips containingspots containing thousands of different nucleic acid-fluorophoresequences, for example, Cy3-labeled DNA sequences, can be prepared usinga robotic microarray spotter and let dry. The glass coverslips can thenbe put to a chamber, for example a chamber shown in the FIG. 7, andanalyzed before exposure to a vapor phase analyte and during or afterexposure to the vapor phase analyte.

The comparison of the before and during and/or after images can be doneelectronically by subtracting the before image from the during and orafter image (for example, FIG. 6). Difference in the intensity of thefluorophore emission patterns in the images indicate that the nucleicacid is responsive to the vapor phase analyte. The difference in theemission pattern may be increase or decrease of the intensity of theemission between the before and during and/or after image. The decreaseof at least about 2%, 5%, 10-15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98% or more and up to 100% is considered as indicative of anucleic acid capable of responding to the odor. The increase in theduring and/or after image of at least about 1%, 3%, 5%, 10%, 20%, 30%,40%, 50%, or more including but not limited to 51-100%, 200%, 300%, and1000%, compared to the before image is considered an increase indicatingthat the nucleic acid is responsive to the analyte in vapor phase.

The sensor responses may be varied by the use of salts or otherchemicals present in the buffer of the nucleic acids, before they aredeposited on the substrate, or different substrates or other during thepreparation of the sensors.

Examples of salts include but are not limited to 50 mM MgCl₂, 50 mMSrCl₂, 50 mM CoCl₂, 50 mM CsCl, 50 mM ZnSO₄, 50 mM UO₂ (NO₃)₂, 50 mMCaCl₂, 50 mM BaCl, 50 mM CrK (SO₄)₂, 50 mM AlCl₃, 50 mM NaCl+10 mMTris+50 μM MgCl₂, 50 mM NaCl+10 mM Tris+50 μM SrCl₂, 50 mM NaCl+10 mMTris+50 μM CoCl₂, 50 mM NaCl+10 mM Tris+50 μM CsCl, 50 mM NaCl+10 mMTris+50 μM ZnSO₄, 50 mM NaCl+10 mM Tris+50 μM UO₂ (NO₃)₂, 50 mM NaCl+10mM Tris+50 μM CaCl₂, 50 mM NaCl+10 mM Tris+50 μM BaCl, 50 mM NaCl+10 mMTris+50 μM CrK (SO₄)₂, 50 mM NaCl+10 mM Tris+50 μM AlCl₃.

Examples of cations useful according to the methods of the presentinvention in testing the optimal conditions for nucleicacid-fluorophores for their responses to vapor phase analytes include,but are not limited to: Ag-based on papers that suggest silver increasesCy3(tm) fluorescence in microarray; Re-based on paper that Rheniumcauses superconducting-like resistance in DNA; transition metals, alsowant to test different oxidation states of the transition metals (Cr,Co, and Zn already tested); Alkali metals, LI, Rb, and Fr (Na, K, and Csalready tested); Alkaline Earth Metals, Be (Mg, Ca, Sr, Ba alreadytested); Lanthanide and Actinide Series, use those which are notpoisonous or radioactive, (U0₂ already used); Groups 3a-6a: use thosewhich have ionic forms soluble in water (Al already used)

The following are anions useful according to the present invention intesting the nucleic acids for their responses to analytes in vaporphase: Cl, NO₃, and SO₄.

Substrates as listed elsewhere in the application, such as differentplastics and surface modified substrates, such as silanized substrates,can modify the response of the nucleic acid to a vapor phase analyte andshould be taken into consideration when testing the nucleic acids andlater when constructing the actual sensing array useful in detectingvapor phase analytes according to the present invention.

EXAMPLE 1

The portable EVID and a schematic overview of the EVID's sensingchamber, sensors, optical components, sniff mechanism, and computercontrol lines are shown in FIG. 1. The EVID uses an array of sensorsthat change their fluorescence intensity upon exposure to brief pulsesof airborne analytes (e.g., “odorants”). The EVID in its present formcontains 16 sensors that can be illuminated and observed at 16 differentexcitation and emission wavelengths. The'sensors are placed along anarrow chamber through which ambient air is drawn (see below). Theoptical elements for illuminating and monitoring the sensors arepositioned along the sides of the chamber (FIG. 1B). Excitation light isproduced by LEDs providing wavelengths appropriate for the sensors beingused (e.g., 460 nm and 530 nm).

Dye-labeled DNA can act as an analyte sensor. As an initial test ofwhether DNA stained with a fluorescent dye responds to analytes, sensorswere constructed from a standard 2.9 kb pBlueScriptSK plasmid mixed withYO-PRO dye (Molecular Probes, Inc.) and dried onto a substrate material(see method details below). Sensors made from YO-PRO alone and rinsedfor 5 min in 70% ethanol showed no analyte responses. (FIG. 2A). Asensor made by mixing a small quantity of plasmid with YO-PRO, however,produced a large and rapid decrease in fluorescence upon exposure topropionic acid, and smaller changes to water, methanol, andtriethylamine (FIG. 2B).

Tests with double-stranded DNA showed no sequence effects. To begintesting whether different sequences of double-stranded DNA per se canproduce sensors of different analyte response profiles, twooligonucleotide oligomers were synthesized that were composed of solelyGC or AT and were designed to form hairpin structures. Althoughdiffering significantly in primary sequence, the two sensors made fromthese hairpins had similar analyte response profiles. The hairpin sensorresponses were also qualitatively similar to the sensors made frompBluescriptSK DNA.

Analyte dilutions as fractions of saturated vapor were: Water, 10⁻¹;methanol (MeOH), 10⁻¹; triethylamine, 10⁻²; and propionic acid, 10⁻¹.Each trace represents the mean of 10 presentations; error bars indicate+/−1 S.D. For experiments with DNA-based analyte sensors, similarmethods were used for each type of sensor. Briefly, DNA in solution wasdiluted to the desired concentration (0.2-40 ng/μl) in TE (10 mM Tris,0.5 mM EDTA). 20 μl of dilute DNA was mixed with 1 μl concentrated dyestock and incubated at room temperature for 5 minutes. Dye-only controlswere made of 1 μl dye stock in 20 μl TE. Sensors were made on asubstrate of acid-washed 16xx silkscreen (10 mm×12 mm). DNA/dye mixtureswere pipetted onto the substrate and allowed to dry for 25 minutes. Eachsensor was rinsed in 70% ethanol for 5 minutes, allowed to dry, thenattached to supports on glass coverslips for testing in the EVID.

Single-stranded DNA sequences can show differential analyte responses.As a further test of whether differences in DNA sequence can producesensors with different response profiles, sensors made fromsingle-stranded DNA stained with the fluorescent dye OliGreen (MolecularProbes, Inc.) were tested. A sensor made from the OliGreen dye aloneshowed a decrease in fluorescence upon exposure to propionic acid, butlittle change with other analytes (not shown). This response was noteliminated with longer rinse times of 10 and 15 min. Sensors made fromOligo dT and oligomer DS003 showed enhanced signals to propionic acidand the other analytes tested (DS003 shown in FIG. 3A). The responseprofiles of these two sensors were similar to each other, and were alsosimilar to the responses of the double-stranded DNA sensors made withYO-PRO (FIG. 2B).

A sensor made with the AJOOI primer sequence, however, had a markedlydifferent analyte response profile (FIG. 3B). This sensor showed anincrease in fluorescence in response to propionic acid and methanol,with relatively little change to the other analytes tested. While otherDNA-based sensors showed responses to propionic acid, none showed asstrong a methanol signal as this AJ001 sensor.

With applied dyes such as OliGreen, there is little control over how thedye interacts with the DNA sequence. In order to define thedye-nucleotide interaction explicitly, we tested oligonucleotides withthe fluorescent dye Cy3(tm) covalently attached to the 5 ′ end duringsynthesis. Sensors made from Cy3(tm)-labeled sequenccs can showdistinctly different analyte response profiles. The LAPP1 sensor (FIG.4A) showed good sensitivity to propionic acid and triethylamine(detection limits at dilutions of about 10⁻³), and less sensitivity tomethanol, DNT and DMMP (detection limits at dilutions of about 2×10⁻²).In contrast, the LAPP2 sensor (FIG. 4B) showed good sensitivity totriethylamine (detection limit at dilutions of about 10⁻³), lesssensitivity to DMMP (detection limit at dilutions of about 2×10⁻²), andno response to propionic acid, methanol, or DNT, even at highconcentration (10⁻¹ dilution).

It is important to emphasize that certain DNA-based sensors, such asLAPP1 shown here, respond to DNT. Besides the nitroaromatic sensingpolymer developed by Dr. Timothy Swager (MIT), no other fluorescentsensor types that we have tested respond to DNT. Additionally, LAPP1responds to DNT at dilutions down to 2×10⁻², or approximately 6 ppb,indicating that these sensors are capable of detecting low vapor-phaseconcentrations of some analytes.

EXAMPLE2

(SEQ ID NO: 1) LAPP1: 5′ GAG TCT GTG GAG GAG GTA GTC 3′ (SEQ ID NO: 2)LAPP2: 5′ CTT CTG TCT TGA TGT TTG TCA ACC 3′ (SEQ ID NO: 3) LAPPAS: 5′TTT GGC TTT CTG GAA ATG GGC 3′ (SEQ ID NO: 4) LAJ001: 5′ ACC AGG ACC TGACTA AGC AGA T 3′

Oligomers LAPP1, LAPP2, LAPPAS, and LAJ001 were synthesized and labeledat the 5′ end with the fluorescent dye Cy3(tm) during synthesis (usingCy3(tm) phosphoramidite from Glen Research). The oligomers were storedin Tris-NaCl (10 mM Tris, 50 mM NaCl, pH 8) at 225 ng/ul, then dilutedto a concentration of 50 ng/ul in distilled waterjust before use.Sensors were constructed by applying 20 ul of dilute oligomer solutionto 10 mm×12 mm pieces of acid-washed 16xx silkscreen. Sensors wereallowed to dry for at least 30 min at room temperature, then attached tosupports for testing.

All sensors were mounted in the device and tested simultaneously. Allwere illuminated with excitation light at 540 nm (30nm bandwidth).Sensors made with LAPP1, LAPPAS, and LAJ001 were observed at 600 nm (10nm bandwidth) and LAPP2 was observed at 610 nm (10 nm bandwidth). Vaporsfrom propionic acid, triethylamine, methanol, DNT, and DMMP (dimethylmethylphosphonate, an organophosphate compound that is a simulant forSarin) were presented to the device using an air dilution olfactometerat the indicated dilutions. For the graphs in the figure, each point ineach curve represents the mean sensor response to ten 2 sec sniffs takenat 30 sec intervals; error bars indicate +/− one standard deviation.Signal amplitudes for the odorants are represented as multiples of thesignal amplitude of background air (indicated by horizontal dashedline).

In the initial tests, the oligomer sequences LAPP1 and LAPP2 showed adistinctly different response profiles to this small test set ofodorants. The LAPP1 sensor showed good sensitivity to propionic acid andtriethylamine (detection limits at dilutions of about 0.001), and lesssensitivity to methanol, DNT and DMMP (detection limits at dilutions ofabout 0.02). In contrast, the LAPP2 sensor showed good sensitivity totriethylamine (detection limit at dilutions of about 0.001), lesssensitivity to DMMP (detection limit at dilutions of about 0.02), andalmost no response to propionic acid, methanol, or DNT, even at highconcentration (0.1 dilution). Sensors made with LAPPAS and LAJ001sequences showed responses similar to LAPP2, but with smalleramplitudes. These data show that sensors that differ only in nucleotidesequence can exhibit different odorant response profiles. LAPP1 respondsto DNT at dilutions down to 0.02, or approximately 6 ppb, indicatingthat these sensors are capable of detecting low vapor-phaseconcentrations.

EXAMPLE 3

The dye-labeled DNA-based sensors described above can be selected usingthe system described herein. The strategy for finding different DNAsequences that respond to different analytes takes advantage of modernhigh-throughput methods and equipment for examining large numbers of DNAinteractions rapidly. An overview of the approach is shown in FIG. 5 andis detailed in the following sections.

Prior to a large-scale sensor screen, details of the steps shown in FIG.5 are established through a series of pilot experiments. The appropriatesequence length is determined, the actual sensor template is designed,and the necessary amplification and labeling conditions are establishedfor generating large numbers of random DNA sequences for use as sensorsusing the methods described elsewhere in the specification. The amountby which the full sequence library needs to be diluted for effectivescreening is also be determined by testing different dilutions.

Determine sensor length. The single-stranded sensors investigated in ourpreliminary studies ranged from 18 to 23 bases in length. The minimumsensor length necessary for differential analyte responses, however, isunknown.

The sensor screen will be most effective if the final sensor libraryrepresents a significant portion of the original sequence library. Thenumber of different sequences in the original. library goes up as4^(no.bases), so a sequence length of 20 bases would yield an originallibrary of approx. 10¹² sequences, far greater than can be screened.

To determine a minimum effective sensor sequence length, LAPP1 and LAPP2sequences labeled with Cy3(tm) are used as described. Sections of thetwo sequences are swapped, beginning with a swap point at the mid-pointof the two sequences. A change in the analyte profile of either originalsequence indicates that an effective sensor sequence is longer than theswap point.

The swap point is moved closer or farther away from the 5′ end (wherethe dye is attached) as necessary until there is no change in theanalyte response profile. That point defines the minimum sensor length.

Design sensor template. Once the minimum sensor length is determined,the sensor template is designed. At least two possible amplificationstrategies are used: polymerase chain reaction (PCR) and primerextension (step 4 in FIG. 5). For PCR amplification, the template willconsist of a random sensor sequence (length determined above) flanked bytwo anchor sequences. Alternatively, primer extension can be used foramplification, where the template will consist of a random sensorportion followed by a single anchor portion (see top of FIG. 5 forschematic representations of templates). In each of these templates, theanchor portion is complementary to the primer sequence(s) to be used forthe amplification (one primer for primer extension, two primers forPCR). Each anchor/primer pair will be short in order to have as littleeffect as possible on the sensor responses, yet must be long enough tohave a sufficiently high melting temperature for the amplificationprocess An anchor/primer sequence of 13 bases will be investigatedinitially, which has an estimated melting temperature of 47° C.

Tests are conducted on both template types to determine the effects offlanking primer portions on analyte responses. Sequences containingknown sensor regions (such as LAPP1 and LAPP2, shown in FIG. 4) flankedby one or two primer sequences will be synthesized along with attcheddye label. The analyte responses of these sensors will be tested usingthe methods described elsewhere. Comparison of the analyte responseswill determine whether PCR or primer extension will be used in theamplification (step 4) and hence will determine final template design.

Determine labeling procedure. Because any post-amplification proceduresfor attaching dye to the sensor sequences must be repeated about 10,000times, the dye to the primer sequence is preferably, but notnecessarily, attached so that it will be incorporated duringamplification. In order to place the dye molecule as close as possibleto the sensor portion of the sequence, the primer is labeled at the 3′end by incorporating an amino-allyl modified dC or dT (Glen Research)during synthesis. N-hydroxysuccinimide functionalized Cy3(tm) (AmershamBiosciences) attaches the dye to the amino-allyl linker. After the dyereaction, a gel filtration purification step removes the unincorporateddye. The dye-labeled primer is then ready to use in the amplificationstep 4.

Determine amplification conditions. As mentioned above, PCR or primerextension is used to amplify samples of the sequence library (step 4 ofFIG. 5), depending on the outcome of the analyte tests described above.

Optimal buffer conditions, reagent concentrations, and thermal cyclingconditions are determined through tests with sensor templates containinga known sensor sequence and using the dye labeled primers describedabove. Amplification is monitored by spotting the amplified sensorsequence onto glass slides and testing for analyte responses using amicroarray scanner.

Determine optimal dilution. The robotic spotter used to spot the senorlibrary onto a microscope slide can apply approximately 10,000 sensorspots. In order to screen the largest possible number of sensorsequences, it is desirable for each spot to contain multiple differentsensor sequences. Too many non-responsive sensor sequences in a spot,however, may obscure the signals from a single responsive sensor.

To estimate the number of non-responsive sequences that could obscure aresponsive sequence, tests are conducted with the known sensor sequencesLAPP1 and LAPP2. As shown in FIG. 4, LAPP1 responds to propionic acidwhereas LAPP2 does not—propionic acid thus discriminates these twosensor sequences. Spots with varying integer ratios of dye-labeled LAPP1and LAPP2 will be applied to microscope slides and exposed to propionicacid under the conditions that is used to screen the entire sensorlibrary. The lowest LAPP1:LAPP2 ratio that still shows a propionic acidsignal discriminable from LAPP2 alone provides an estimate of the numberof sensor sequences per spot. This number of sensors per spot will thenbe set by the dilution in step 2 and the sample volume used in step 3(FIG. 5).

Create a large-scale random library of different oligonucleotide-basedsensors. The steps outlined in FIG. 5 and detailed above are followed togenerate and amplify a sensor library, which is spotted onto microscopecoverslips for screening with analytes.

A random sequence library is synthesized using the sensor templatedescribed above (step 1 of FIG. 5). The sequence library is diluted sothat 1 μl samples contain at least one, and possibly multiple, differentsequences (step 2). The 1 μl samples are put into 96-well plates (step3).

Primer, bases, polymerase enzyme, and buffer are then added to eachwell. Four plates at a time (384 samples) are amplified using a PCT-225PCR Tetrad thermal cycler (step 4). It is estimated that four plates canbe amplified in a day, so amplification of the full 10,000 sample sensorlibrary will require 26 days. A BioRobotics MicroGrid II microarrayspotter is then used to produce sensor slides containing 10,000 spots ofthe amplified sequences (step 5). Multiple replicates of the spottedslides are generated, most of which are stored for future screenings.

The sensor library generated using the methods described above arescreened with a set of explosive-related compounds and CWA simulants.Sensor spots showing responses to these compounds are further diluted,amplified, and spotted onto slides for additional testing with theanalytes to locate the unique sensor sequences. The sequences are thendetermined using standard DNA sequencing methods.

A Packard BioChip Technologies ScanArray 4000 array scanner is used toscan a sensor slide while exposing it to analytes of interest. Thisrequires the construction of a sealed slide holder so that analytes canbe applied to the spots during scanning. One exemplary design is shownon FIG. 7. Examples of the analytes that can be used in the screeningare shown in Tables 3-5.

Spots showing a change in fluorescence with analyte exposure areexamined further. The amplified samples that produced the spots arefurther diluted to produce sub-samples containing individual sequences.These sub-samples are amplified again using the same amplificationprotocols developed above and spotted onto slides. The spots on theseslides therefore contain individual sensor sequences. These slides aretested with analytes in the array scanner to identify individual sensorsequences for the odors of interest. The sequences in the sub-samplesthat produced the responsive sensors are determined using standard DNAsequencing methods. The final appropriate sequences are thensynthesized, labeled, and tested directly in the EVID.

For each of the sensors identified in the sensor library screendescribed above, concentration-response functions are determined foreach of the explosives, related compounds, and chemical agents listed inTable 3, 4 and 5. Using these data for individual sensors, an optimizedsensor array is constructed and the lower detection limit for each ofthe compounds determined. These detection limits are compared to thereported sensitivities of commercially available devices, totoxicological data for the chemical agents, and, where data areavailable, to the behavioral thresholds of trained dogs. TABLE 3 ExampleExplosives Target Related compounds Function Reference C-4 DimethylTaggant dinitrobutane Dioctyl Sebacate Plasticizer 2-ethyl-1-hexanolSolvent Toluene Solvent Ron Ray, CDRI, pers. comm. RDX CyclohexanoneSolvent Jenkins and O'Reilly (1974) TNT DNT Synthetic precursorDinitrobenzene Contaminant (DNB)

TABLE 4 Example Chemical Warfare Agents/Blister Agents Target Relatedcompounds Function Reference Mustard (H) Dibutyl sulfide Simulant Pal etal. (1993) 2-Chloroethyl Simulant Jaeger et al. (1999) phenyl sulfide

TABLE 5 Example Chemical Warfare Agents/Nerve Agents Target Relatedcompounds Function Reference Sarin (GB) Diisopropyl Simulant Pal et al.(1993) methylphosphonate (DIMP) Dimethyl Synthetic methylphosphonateprecursor (DMMP) Isopropyl Degradation * methylphosphonate product(IMPA) Triethyl phosphite Simulant Soman (GD) Triethyl phosphateSimulant * VX Tributyl phosphate Simulant * DMMP Simulant Pal et al.(1993)* Fromhttp://www.dean.usma.edu/chem/Faculty/fountain/Fountainprcrsrch.htm

An air-dilution olfactometer, based on standard olfactometry conceptsand modeled after a system used in dog studies (Hartell et al., 1998),is used to deliver controlled dilutions 6f analytes to the EVID. In thepresent configuration, filtered compressed air is fed to a bank ofmass-flow controllers (Teledyne Hastings Instruments) to set flow ratesthrough four air or analyte channels. Eight channels can also be used.One channel sets the background (diluent) air flow from 1 L/min to 10L/min. The other three channels feed the air (flow rates from 10 ml/minto 10 L/min) through gas-washing bottles and other custom glasswarecontaining analyte samples. Downstream of the analyte vessels, theanalyte stream in each channel is controlled by electric valves (KIPInc.), directing flow to exhaust or to a manifold bringing all channelsback together. The manifold is connect to a glass analyte port, intowhich the snout of the EVID is placed for sampling. Analyte dilutionsare determined by the relative flow rates through the diluent airchannel and the analyte channel. Total flow rate is typically 10 L/min.

Sensors are characterized by their responses to the analytes over arange of concentrations. To collect concentration-response data for eachanalyte, a concentration series is delivered to the EVID using theolfactometer described above. An ascending series of concentrations arepresented and ten sniffs at each concentration taken, with 30 secbetween sniffs. Each concentration series starts with a dilution of5×10⁻⁴ of saturated vapor concentration and ascend, in steps thatapproximately double the concentration, to a dilution of 10⁻¹ (i.e.,5×10⁻⁴, 10⁻³, 2×10⁻³, 5×10⁻³, 10⁻², 2×10⁻², 5×10⁻³, 10⁻¹). Data from thedevice is logged to a separate computer for analysis and display.

The detection limit for each sensor is determined by comparing theamplitude of the signal to that of clean air. The lowest concentrationof an analyte that elicits a signal that is significantly different fromthe clean air signal is the detection limit of the sensor for thatsimulant. Detection limits for all the sensors available for all theanalytes are determined in this way.

An optimized array of sensors, each with a low detection limit for oneor more analytes, is selected. The detection limits of this array foreach compound are determined using random presentations of the analytesat the dilutions listed above. Performance of the array is evaluatedusing signal detection theory and ROC curves. To evaluate the detectionlimits of the EVID determined in these studies, the values are comparedto CWA toxicological data and to the sensitivities of other detectiondevices, including trained dogs.

EXAMPLE 4

Preparation and testing of nucleic acids for their responsiveness toanalytes in vapor phase.

DNA-Cy3(tm) Sensor Library. A set of DNA oligomers with random internalsequence and fixed ends (see FIG. 11) have been prepared by the TuftsUniversity DNA/Protein Core Facility. Random nucleotide incorporationwas used to generate the random portion of the oligomer. This random setprovides DNA templates for amplification and labeling to produce aDNA-Cy3(tm) library for screening as described below.

Two methods are possible for initial amplification of the sensorlibrary:

PCR. Polymerase chain reaction can be used to initially amplify thelibrary sequences. The random oligomer library with determined primerand anchor sequences can be serially diluted, with the last dilutionbeing no greater than 1:10, until it is calculated that each well in amicrotitre plate contains, on average, one molecule of DNA. The DNAmolecule(s) in each well is then be amplified and labeled by PCR throughthe reaction described below.

Bacteria A preliminary library has been constructed by putting therandom sequences of DNA (FIG. 11) into plasmids using one of thecommercially available single copy cloning kits (TOPO TA or Zero BluntTOPO PCR) from Invitrogen. Plasmids were transformed into E. coli andcolonies selected for expression of antibiotic resistance genes carriedon the plasmid. The selection of bacteria with a drug resistant singlecopy plasmid allows one and only one DNA sensor sequence to be expressedin each colony. Colonies can be picked and grown in 96-well plates.After growth, a small portion of the bacteria can be lysed and thesensor sequence amplified and Cy3(tm) labeled in the wells using theprimers as described below.

DNA Labeling Procedure. DNA can be fluorescently labeled using a varietyof mechanisms and dyes. We have chosen Cy3(tm) to facilitate the use ofexisting microarray technology in our experiments. Importantly, ourpreliminary studies show that 5′ labeled single-stranded DNA-Cy3(tm)sensors respond robustly to odors. One method of 5′ Cy3(tm) labeling ofDNA uses a 5′ capping reaction involving phosphoramidite chemistryduring oligomer synthesis (reagents available from Glen Research).

Although phosphoramidite chemistry provides a convenient method forattaching Cy3(tm) to DNA, it can only be used in synthesis reactions.This does not allow for attaching Cy3(tm) during amplification of theDNA sensor sequences. We have instead chosen to use a Cy3(tm) labeledprimer in the PCR reaction to allow labeling at a defined location (FIG.12). Cy3(tm)-labeled primer produced by first synthesizing an oligomerwith a modified thymidine, Amino-modifier C2dT (Glen Research), at the3′ end. C2dT was selected as an attachment site for Cy3(tm) because itsshort 2-carbon linking group gives the greatest possibility forelectrochemical interactions between the DNA and Cy3(tm) attached to thetwo-carbon linker. Cy3(tm)-NHS ester (Amersham) was attached via anamide bond to the C2dT, using the NHS ester as a leaving group in thereaction (Amersham protocols were followed for this reaction). Theprimer was 3′ labeled so that the final DNA-Cy3(tm) product is 5′labeled after removing all but the labeled base of the primer sequenceusing the restriction enzyme BsrD1. Results using Cy3(tm) linked todefined oligomers via a 5′ C2dT show the effectiveness of this method increating DNA-Cy3(tm) odor sensors.

In other PCR studies, Taq polymerase has been used to add Cy3(tm)-linkednucleotides to a growing strand of DNA without interrupting the growthof the strand. Our method is similar, except that the label is locatedin the primer at the beginning of the growing strand instead ofinterspersed as Taq randomly adds labeled nucleotides. To removeunlabeled primer, which could exist if the reaction to attach Cy3(tm) tothe primer is incomplete, Cy3(tm)-labeled primer was purified byreverse. phase high-pressure liquid chromatography (RP-HPLC).Preliminary results using this purification technique were successfuland will be optimized.

Using the computer program Oligo no likely primer secondary structuresor dimers were predicted, but PCR conditions need to be optimized formaximal amplification. Specific factors to optimize are MgCl₂concentration, annealing temperature, and primer concentration.Optimization of the PCR is carried out with known sequences prior topreparation of the random library, but preliminary data has shown that aCy3(tm) labeled PCR product of the appropriate size can be producedusing our labeled primer. PCR products will be analyzed using 19%polyacrylamide gel electrophoresis. DNA bands will be examined usingSBYR to show single-stranded DNA bands, and the Cy3(tm) fluorescent tagitself to show labeled bands.

The anchor sequence is removed by using a restriction enzyme, BsrD1 (NewEngland Biolabs) to cut the anchor sequence from the label.

Although there are sticky ends remaining after BsrD1 digestion, themelting of the complementary strands in the purification process is morethan adequate to separate the sticky ends. There is theoretically someloss in library yield due to random sequences which contain either theBsD1 restriction site or the primer sequence. Loss due to a BsrD1restriction site in the random sequence is calculated to be 13×4¹²sequences. Loss due to random matching of the primer sequence iscalculated to be 45 exact matches for each primer in each orientation or46 total sequences lost. These losses combined represent less than 0.02%of the total library.

Purification of PCR Product. For method optimization, oligo dT spincolumns (Amersham) are used to purify DNA. Polyacrylimide gelelectrophoresis is used to analyze products. However, for a large DNAlibrary, these methods are too slow and labor intensive. Separatinglabeled single-stranded DNA (ssDNA) from the complementary strand,primers, unused dNTPs, and reaction buffers is accomplished throughwashing after the desired ssDNA is bound to a substrate. Two possiblemethods are as follows:

Micro-titre plates with solid phase oligo dT in the wells are availabefrom Sequitur, Inc. Hot DNA from the PCR reaction is placed in the wellsand as cooling occurs, the poly(A) tail of the labeled DNA anneals withthe oligo dT in the well. The rest of the PCR reaction mixture is thenwashed away.

Using TdT, a biotin conjugated base is added to the 3′ end of thelabeled sequence. This is then bound to a strepavidin coated slide andthe remaining material washed away.

Microarray Slide Production. Coverslips containing spots of thousands ofdifferent DNA-Cy3(tm) sequences are created using a robotic microarrayspotter. Coverslips are then placed in a specially constructed chamber(FIG. 7).

Preliminary tests have been conducted using a prototype version of thischamber. Spots of 12 different sequences labeled with Cy3(tm) duringsynthesis were spotted onto a coverslip using a spotting robot(BioRobotics MicroGrid II), and the coverslip placed in the prototypechamber. Odors were delivered to the chamber in a controlled mannerusing a syringe pump. The coverslip was imaged using a microarrayscanner (ScanArray 4000) before and after odor delivery.

Additional Results. The results outlined here indicate possible ways ofmodifying sensor responses by altering the salt content of theDNA-Cy3(tm) buffer during sensor construction and by using differentsubstrates for making the sensors.

Effects of Different Salts. We have found that the salt content of theDNA-Cy3(tm) solution used to make the sensor can have an effect onsensor responses, both in terms of amplitude and in odor responseprofile.

The following salts were tested: 50 mM MgCl₂, 50 mM SrCl₂, 50 mM CoCl₂,50 mM CsCl, 50 mM ZnSO₄, 50 mM UO₂ (NO₃ )₂, 50 mM CaCl₂, 50 mM BaCl, 50mM CrK (SO₄)₂, 50 mM AlCl₃, 50 mM NaCl+10 mM Tris+50′ M MgCl₂, 50 mMNaCl+10 mM Tris+50 μM SrCl₂, mM NaCl+10 mM Tris+50 μM CoCl₂, 50 mMNaCl+10 mM Tris+50 μCsCl, 50 mM NaCl+10 mM Tris+50 μM ZnSO₄, 50 mMNaCl+10 mM Tris+50 μM UO₂ (NO₃ )₂, 50 mM NaCl+10 mM Tris+50 μM CaCl₂, 50mM NaCl+10 mM Tris+50 μM BaCl, 50 mM NaCl+10 mM Tris+50 μM CrK (SO₄)₂,50 mM NaCl+10 mM Tris+50 μM AlCl₃. In addition to these saltcombinations, DNA-Cy3(tm) solutions tested contained 500 μM sodiumborate buffer.

In these experiments, the indicated salt was added to the DNA-Cy3(tm)solution that was then applied to the substrate and dried. 20 μl ofsolution was applied to a piece of 10×12 mm silkscreen. After drying,the actual concentration of salt on the sensor is unknown, but isestimated to be much higher.

The following anions are also useful in testing the nucleic acids fortheir responses to analytes in vapor phase: Cl (already used in ourtests); NO₃ (already used); SO₄ (already used).

The references cited herein and throughout the specification are hereinincorporated by reference in their entirety. The examples above, aremeant to provide guidance in making and using the present invention,however, the invention is meant to cover all the equivalents of thesepreferred embodiments which one skilled in the art is capable ofpreparing based upon this disclosure.

REFERENCES

-   Aathithan, S., Plant, J. C., Chaudry, A. N., and French, G. L.    (2001). Diagnosis of bacteriuria by detection of volatile organic    components in urine using an automated headspace analyzer with    multiple conducting polymer sensors. J. Clin. Microbiol.,    39:2590-2593.-   Alkasab, T. K, Bozza, T. C., Cleland, T. A., Dorries, K. M.,    Pearce, T. C., White, J., and Kauer, J. S. (1999). Characterizing    complex chemosensors: Information theoretic analysis of olfactory    systems. Trends Neurosci., 22:102-108.-   Alkasab, T. K., White, J., and Kauer, J. S. (2002). A computational    system for simulating and analyzing arrays of biological and    artificial chemical sensors. Chem. Senses, 27:261-275.-   Bartlett, P. N. and Gardner, J. W. (1992). Odour sensors for an    electronic nose. In Gardner, J. W. and Barlett, P. N., editors,    Sensors and Sensory Systems for an Electronic Nose, pages 31-51.    Kluwer Academic Publishers, Dordrecht, Netherlands.-   Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H., and    Toole, J. J. (1992). Selection of single-stranded DNA molecules that    bind and inhibit human thrombin. Nature, 355:564-566.-   Bouche, M. P., Lambert, W. E., Bocxlaer, J. F. V., Piette, M. H.,    and Leenheer, A. P. D. (2001). Quantitative determination of    n-propane, iso-butane, and n-butane by headspace GC-MS in    intoxications byinhalation of lighter fluid. J. Anal. Toxicol.,    26:35-42.-   Buck, L. and Axel, R. (1991). A novel multigene family may encode    odorant receptors: A molecular basis forodor recognition. Cell,    65:175-187.-   Cam, D. and Gagni, S. (2001). Determination of petroleum    hydrocarbons in contaminated soils using solid-phase microextraction    with gas chromatography-mass spectroscopy. J. Chromatogr. Sci.,    39:481-486.-   Cancho, B., Ventura, F., and Galceran, M. T. (2002). Determination    of aldehydes in drinking water usingpentafluorobenzylhydroxylamine    derivatization and solid-phase microextraction. J. Chromatogr.,    943:1-13.-   Chen, S., Mahadevan, V., and Zieve, L. (1970). Volatile fatty acids    in the breath of patients with cirrhosis oftheliver. J. Lab. Clin.    Med., 75:622-627.-   Christensen, T. A. and White, J. (2000). Representation of olfactory    information in the brain. In Finger, T. E., Silver, W. L., and    Restrepo, D., editors, Neurobiology of Taste and Smell, pages    201-232. John Wiley & Sons, New York.-   Dickinson, T. A., White, J., Kauer, J. S., and Walt, D. R. (1998).    Current trends in ‘artificial-nose’ technology. Trends Biotechnol.,    16:250-258.-   Ellington, A. D. and Szostak, J. W. (1990). In vitro selection of    RNA molecules that bind specific ligands. Nature, 346:818-822.-   Freund, M. S. and Lewis, N. S. (1995). A chemically diverse    conducting polymer-based “electronic nose”. Proc. Nat. Acad. Sci.    USA, 92:2652-2656.-   Gardner, J. W. and Barlett, P. N. (1994). A brief history of    electronic noses. Sensors and Actuators B, 18-19:211-220.-   Gardner, J. W. and Bartlett, P. N., editors (1992). Sensors and    sensory systems for an electronic nose, volume 0. Kluwer Academic    Publishers, Dordrecht, The Netherlands.-   Gardner, J. W. and Hines, E. L. (1997). Pattern analysis techniques.    In Kress-Rogers, E., editor, Handbook of Biosensors and Electronic    Noses: Medicine, Food, and the Environment, pages 633-652. CRC    Press, BocaRaton, Fla.-   Gelmont, D., Stein, R. A., and Mead, J. F. (1981). Isoprene—the main    hydrocarbon in human breath. Biochem. Biophys. Res. Commun.,    99:1456-1460.-   George, V., Jenkins, T., Leggett, D., Cragin, J., Phelan, J., Oxley,    J., and Pennington, J. (1999). Progress ondete mining the vapor    signature of a buried landmine. In Proceedings of the 13th Annual    International Symposium on Aerospace/Defense Sensing, Simulation, &    Controls, pages 258-269.-   George, V., Jenkins, T., Phelan, J., Leggett, D., Oxley, J., Webb,    S., Miyares, P., Cragin, J., Smith, J., andBerry, T. (2000).    Progress on determining the vapor signature of a buried landmine. In    Proceedings of the 14th Annual Interndtional Synposium on    Aerospace/Defense Sensing, Simulation, & Controls.-   Grate, J. W., Rose-Pehrsson, S. L., Venezky, D. L., Klusty, M., and    Wohltjen, H. (1993). Smart sensor system for trace organophosphorus    and organosulfur vapor detection employing a temperature-controlled    arrayof surface acoustic wave sensors, automated sample    preconcentration, and pattern recognition. Anal. Chem.,    65:1868-1881.-   Grote, C. and Pawliszyn, J. (1997). Solid-phase microextraction for    the analysis of human breath. Anal. Chem., 69:587-596.-   Hamaguchi, N., Ellington, A., and Stanton, M. (2001); Aptamer    beacons for the direct detction of proteins. Anal. Biochem.,    294:126-131.-   Hartell, M., Myers, L., Waggoner, L., Hallowell, S., and    Petrousky, J. (1998). Design and testing of a quantitative vapor    delivery system. In Proceedings of the 5th International Symposium    on the Analysis and Detection of Explosives, Washington, D.C.:    Treasurey Department.-   Jayasena, S. D. (1999). Aptamers: An emerging class of molecules    that rival antibodies in diagnostics. Clin. Chen., 45:1628-1650.-   Jenkins, T. F., Leggett, D. C., Miyares, P. H., Walsh, M. E.,    Ranney, T. A., Cragin, J. H., and George, V.(2001). Chemical    signatures of TNT-filled land mines. Talanta, 54:501-513.-   Jenkins, T. F., Walsh, M. E., Miyares, P. H., Kopczynski, J. A.,    Ranney, T. A., George, V., Pennington, J. C., and T. E. Berry, J.    (2000). Analysis of explosives-related chemical signatures in soil    samples collected near buried land mines. ERDC TR-00-5, CRREL.-   Jhaveri, S., Rajendran, M., and Ellington, A. D. (2000a). In vitro    selection of signaling aptamers. Nature Biotech., 18:1293-1297.-   Jhaveri, S. D., Kirby, R., Conrad, R., Maglott, E. J., Bowser, M.,    Kennedy, R. T., Glick, G., and Ellington, A. D. (2000b). Designed    signaling aptamers that transduce molecular recognition to changes    influorescence intensity. J. Am. Chem. Soc., 122:2469-2473.-   Kauer, J. S. (1987). Coding in the olfactory system. In    Finger, T. E. and Silver, W. L., editors, Neurobiology of Taste and    Smell, pages 205-231. John Wiley & Sons, Inc, New York.-   Kauer, J. S. (1991). Contributions of topography and parallel    processing to odor coding in the vertebrate olfactory pathway.    Trends Neurosci, 14:79-85.-   Kauer, J. S. and White, J. (2002). Representation of odor    information in the olfactory system: from biology to an artificial    nose. In Barth, F. G., Humphrey, J. A. C., and Secomb, T. W.,    editors, Sensors and Sensing in Biology and Engineering.    Springer-Verlag, Berlin.-   Kent, P. F. and Mozell, M. M. (1992). The recording of    odorant-induced mucosal activity patterns with avoltage-sensitive    dye. J Neurophysiol, 68:1804-1819.-   Kundu, S. K., Bruzek, J. A., Nair, R., and Judilla, A. M. (1993).    Breath acetone analyzer: Diagnostic toolto monitor dietary fat loss.    Clin. Chem., 39:87-92.-   Lambropoulou, D. A. and Albanis, T. A. (2001). Optimization of    headspace solid-phase microextraction conditions for the    determination of organophosphorus insecticides in natural waters. J.    Chromatogr., 922:243-255.-   Leggett, D. C., Cragin, J. H., Jenkins, T. F., and Ranney, T.    (2001). Release of explosive-related vapors fromland mines. ERDC    TR-01 6, CRREL.-   Lu, X.-C. M., Slotnick, B. M., and Silberberg, A. M. (1993). Odor    matching and odor memory in the rat. Physiol. Behav., 53:795-804.-   MacKay-Sim, A., Shaman, P., and Moulton, D. G. (1982). Topographic    coding of olfactory quality: Odorant-specific patterns of epithelial    responsivity in the salamander. J. Neurophysiol., 48:584-596.-   Mendis, S., Sobotka, P. A., and Euler, D. E. (1994). Pentane and    isoprene in expired air from humans: Gas-chromatographic analysis of    single breath. Clin. Chem., 40:1485-1488.-   Mitchell, S., Ayesh, R., Barrett, T., and Smith, R. (1999).    Trimethylamine and Foetor Hepaticus. Scand. J. Gastroenterol,    34:524-528.-   Moore, D. (2001). Preparation and analysis of dna. In Ausubel, F.    M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G.,    Smith, J. A., and Struhl, K., editors, Current Protocols in    Molecular Biology, volume 1, chapter 2. John Wiley & Sons, Inc.-   Mosaddegh, M. H., Richardson, R:, Stoddart, R. W., and McClure, J.    (2001). Application of solid-phase micro-extraction technology to    drug screening and identification. Ann. Clin. Biochem., 38:541-547.-   Musshoff, F., Junker, H., and Madea, B. (2002). Simple determination    of 22 organophosphorous pesticides in human blood using headspace    solid-phase microextraction and gas chromatography with    massspectrometric detection. J. Chromatogr. Sci., 40:29-34.-   Narasimhan, L. R., Goodman, W., and Patel, C. K. N. (2001).    Correlation of breath ammonia with blood ureanitrogen and creatinine    during hemodialysis. Proc. Natl. Acad. Sci., 98:4617-4621.-   Park, J., Groves, W. A., and Zellers, E. T. (1999). Vapor    recognition with small arrays of polymer-coated microsensors. a    comprehensive analysis. Anal. Chem., 71:3877-3886.-   Passe, D. H. and Walker, J. C. (1985). Odor psychophysics in    vertebrates. Neurosci. Biobehav. Rev., 9:431-467.-   Persaud, K. and Dodd, G. (1982). Analysis of discrimination    mechanisms in the mammalian olfactory system using a model nose.    Nature, 299:352-355.-   Phillips, M., Gleeson, K., Hughes, J. M. B., Greenberg, J.,    Cataneo, R. N., Baker, L., and McVay, W. P. (1999). Volatile organic    compounds in breath as markers of lung cancer: a cross-sectional    study. Lancel, 353:1930-1933.-   Ping, W., Yi, T., Haibao, X., and Farong, S. (1997). A novel method    for diabetes diagnosis based on electronic nose. Biosens.    Bioelectron., 12:1031-1036.-   Potyrailo, R. A., Conrad, R. C., Ellington, A. D., and    Hieftje, G. M. (1998). Adapting selected nucleic acidligands    (aptamers) to biosensors. Anal. Chem., 70:3419-3425.-   Sassanfar, M. and Szostak, J. W. (1993). An RNA motif that binds    ATP. Nature, 364:550-553.-   Schneider, J. F., Boparai, A. S., and Reed, L. L. (2001). Screening    for Sarin in air and water by solid-phase microextraction-gas    chromatography-mass spectrometry. J. Chromatogr. Sci., 39:420-424.-   Severin, E. J., Doleman, B. J., and Lewis, N. S. (2000). An    investigation of the concentration dependence andresponse to analyte    mixtures of carbon black/insulating organic polymer composite vapor    detectors. Anal. Chem., 72:658-668.-   Sidebothiam, R. L. and Baron, J. H. (1990). Hypothesis: Helicobacter    pylori, urease, mucus, and gastric ulcer. Lancet, 335:193-195.-   Slotnick, B. M., Kufera, A., and Silberberg, A. M. (1991). Olfactory    learning and odor memory in the rat. Physiol. Behav., 50:555-561.-   Tangerman, A., Meuwese-Arends, M. T., and Jansen, J. B. M. J.    (1994). Cause and composition of foetor hepaticus. Lancet, 343:483.-   Tassopoulos, C. N., Barnett, D., and Fraser, T. R. (1969).    Breath-acetone and bloqd-sugar measurements indiabetes. Lancet,    1(7609):1282-1286.-   Tuerk, C. and Gold, L. (1990). Systematic. evolution of ligands by    exponential enrichment: RNA ligands tobacteriophage T4 DNA    polymerase. Science, 249:505-510.-   Tyagi, S. and Kramer, F. R. (1996). Molecular beacons: Probes that    fluoresce on hybridization. Nature Biotech., 14:303-308.-   White, J., Dickinson, T. A., Walt, D. R., and Kauer, J. S. (1998).    An olfactory neuronal network for vaporrecognition in an artificial    nose. Biol. Cybern., 78:245-251.-   White, J. and Kauer, J. S. (1999). Odor recognition in an artificial    nose by spatio-temporal processing usingan olfactory neuronal    network. Neurocomputing, 26-27:919-924.-   White, J., Kauer, J. S., Dickinson, T. A., and Walt, D. R. (1996).    Rapid analyte recognition in a device basedon optical sensors and    the olfactory system. Anal. Chem., 68:2191-2202.-   Yamazaki, K., Singer, A., and Beauchamp, G. K. (1998-1999). Origin,    functions and chemistry of h-2 regulated odorants. Genetica,    104:235-240.-   Yang, J. S. and Swager, T. M. (1998). Porous shape persistent    fluorescent polymer films: An approach toTNT sensory materials. J.    Am. Chem. Soc., 120:5321-5322.-   Youngentob, S. L., Markert, L. M., Mozell, M. M., and Hornung, D. E.    (1990). A method for establishing afiveodor identification confusion    matrix task in rats. Physiol. Behav., 47:1053-1059.-   Zellers, E. T., Batterman, S. A., Han, M., and Patrash, S. J.    (1995). Optimal coating selection for the analysis of organic vapor    mixtures with polymer-coated surface acoustic wave sensor arrays.    Anal. Chem., 67:1092-1106.-   Zhang, Z., Yang, M. J., and Pawliszyn, J. (1994). Solid-phase    microextraction. Anal. Chem., 66:844A-853A.

1. A method for detecting an analyte in an air sample comprising thesteps of: a. contacting said air sample with a nucleicacid/fluorophore-based sensor array comprising a substrate; and anucleic acid labeled with a fluorophore dispersed on said substrate,said nucleic acid labeled with a fluorophore providing a characteristicoptical response when subjected to excitation light energy in thepresence of the analyte; and b. detecting the presence or absence ofsaid analyte.
 2. The method of claim 1, wherein said nucleic acid isdispersed on a plurality of internal and external surfaces saidsubstrate.
 3. The method of claim 1, wherein said contacting furthercomprises drawing an air sample believed to contain said analyte into asample chamber and exposing said nucleic acid/fluorophore based sensorarray to said air sample.
 4. The method of claim 1, wherein saiddetecting further comprises: a. illuminating said nucleicacid/fluorophore based sensor array with excitation light energy; and b.measuring an optical response produced by said nucleic acid/fluorophorebased sensor array due to the presence of said analyte with a detectormeans.
 5. The method of claim 4, further comprising identifying saidanalyte by employing a pattern-matching algorithm; and comparing saidoptical response of said nucleic acid/fluorophore based sensor arraywith said characteristic optical response.
 6. The method of claim 4,further comprising identifying said analyte by providing spatio-temporalresponse patterns of said optical response; and recognizing saidpatterns through a method selected from the group consisting of atemplate matching, neural networks, delay line neural networks, andstatistical analysis.
 7. The method of claim 1, wherein the air sampleis suspected of containing explosive materials.
 8. The method of claim1, wherein the air sample is suspected of containing a chemical weaponsagent.
 9. A method of selecting a nucleic acids capable of responding toa vapor phase analyte, said method comprising: a. contacting the nucleicacid labeled with a fluorophore with an analyte in vapor phase; and b.measuring the emission proflile of the fluorophore in the presence andabsence of the target analyte, wherein a difference in the emissionprofile indicates that the nucleic acid is responsive to the analyte invapor phase.
 10. The method of claims 1, 4 and 9, wherein the nucleicacid is 1-3000 bases long.
 11. The method of claims 1, 4 and 9, whereinthe nucleic acid is 10-500 bases long.
 12. The method of claims 1, 4 and9, wherein the nucleic acid is 15-24 bases long.
 13. The method ofclaims 1, 4 and 9, wherein the fluorophore is attached to the 3′ regionor a 5′ region of the nucleic acid.
 14. The method of claims 1, 4 and 9,where the nucleic acid is internally labeled with the fluorophore. 15.The method of claim 14, wherein the fluorophore is an applied dye. 16.The method of claim 15, wherein the applied dye is YO-PRO or OliGreen.17. The method of claims 1, 4 and 9, wherein the substrate is a silkscreen.
 18. The method of claims 1 4 and 9, wherein the substrate isglass.
 19. A sensing system for detecting and identifying a volatilecompound in an air sample comprising: a. a nucleic acid/fluorophorebased sensor array comprising a plurality of nucleic acids; b. afluorophore attached to said nucleic acids; c. a plurality of substrateswherein said nucleic acids are attached to; d. a substrate support; e.an excitation light source array comprising a plurality of light sourcesoptically coupled to said sensor elements; f. a detector arraycomprising a plurality of detectors optically coupled to said sensorelements; g. a sample chamber for housing said sensor elements, saidlight source array, said detector array; h. a sampling means enclosed insaid chamber for drawing said ambient air into said chamber for contactwith said nucleic acid/fluorophore based sensor array for a controlledexposure time; i. a controller means in electrical communication withsaid light sources, said detectors, and said sampling means, saidcontroller means electrically coordinating and switching said samplingmeans with said light sources and said detectors for sampling saidambient air, measuring optical responses of said nucleicacid/fluorophore based sensor arrays to said ambient air sample, anddetecting said volatile compound; and j. an analyte identificationalgorithm for comparing said measured sensor optical responses tocharacteristic optical responses of said sensors to target analytes andidentifying said analyte in said air sample.
 20. A sensing system forintelligent detecting and identifying an analyte in an air samplecomprising: a. a nucleic acid/fluorophore based sensor array comprisinga plurality of nucleic acids attached to a fluorophore; b. a detectorarray comprising a plurality of detectors in communication with saidnucleic acid/fluorophore based sensor array; c. a sampling chamber forhousing said nucleic acid/fluorophore based sensor array and saiddetector array; d. a sampling means enclosed in said chamber for drawingsaid ambient air into said chamber for contact with saidnucleicacid/fluorophore based sensor array for a controlled exposuretime; e. a microcontroller in communication with said sampling means andsaid detector array, said controller means coordinating and switchingsaid sampling means and said detector array for sampling said ambientair, measuring responses of said sensors to said air sample, detectingsaid analyte, and reporting an analyte detection result; f. a samplingalgorithm for directing said microcontroller; and g. an analyteidentification algorithm in communication with said sampling algorithmand said microcontroller, said identification algorithm comparing saidmeasured sensor optical responses before and after exposure to theanalyte to characteristic responses of said sensors to analytes andidentifying said analyte in said air sample.
 21. The sensing system ofclaim 20, wherein said identification algorithm comprises a responsereport comparing a spatio-temporal pattern of said measured opticalresponses to a spatio-temporal pattern of said characteristic responses;and an identification report selected from the group consisting of apattern match, a delay line neural network match, and a neuronal networkmatch.
 22. The sensing system of claim 20, wherein the sensing system isattached to a shipping container.
 23. The sensing system of claim 20,wherein the sensing system is attached to an x-ray screening machine.24. The sensing system of claim 20, wherein the sensing system isremotely controllable.
 25. The sensing system of claim 20, wherein thesensing system is incorporated into. a hand-held device.
 26. A sensorarray system for remote characterization of a gaseous or vapor, sample,comprising: a. a plurality of sensors, wherein at least one sensorcomprises nucleic acid/fluorophore combination comprising a plurality ofnucleic acids attached to a fluorophore, wherein the plurality ofsensors provide -a detectable signal when contacted by an analyte; b. ameasuring apparatus, in communication with plurality of sensors capableof measuring the detectable signal; c. a transmitting device, incommunication with the measuring apparatus for transmitting informationcorresponding to the detectable signal to a remote location via theInternet, fiber optic cable, and/or an air-wave frequency; and acomputer comprising a resident algorithm capable of characterizing theanalyte.
 27. The sensor array system according to claim 23, wherein thesensor system comprises a plurality of measuring apparatuses.